Methods of cell culture for adoptive cell therapy

ABSTRACT

An improved method of culturing cells for cell therapy applications that includes growing desired cells in the presence of antigen-presenting cells and/or feeder cells and with medium volume to surface area ratio of up to 1 ml/cm 2  if the growth surface is not comprised of gas permeable material and up to 2 ml/cm 2  if the growth surface is comprised of gas permeable material. The desired cells are at a surface density of less than 0.5×10 6  cells/cm 2  at the onset of a production cycle, and the surface density of the desired cells plus the surface density of the antigen presenting cells and/or feeder cells are at least about 1.25×10 5  cells/cm 2 .

RELATED APPLICATION

The present application is a divisional of U.S. patent application Ser.No. 15/395,662, entitled “METHODS OF CELL CULTURE FOR ADOPTIVE CELLTHERAPY”, filed Dec. 30, 2016 which is a continuation of U.S. patentapplication Ser. No. 14/579,373, now U.S. Pat. No. 9,567,565, entitled“IMPROVED METHODS OF CELL CULTURE FOR ADOPTIVE CELL THERAPY”, filed Dec.22, 2014, which is a divisional of U.S. patent application Ser. No.13/475,700, now U.S. Pat. No. 8,956,860 entitled “IMPROVED METHODS OFCELL CULTURE FOR ADOPTIVE CELL THERAPY,”, filed May 18, 2012, which is acontinuation-in-part of U.S. Pat. No. 12/963,597, filed Dec. 8, 2010,now U.S. Pat. No. 8,809,050, issued Aug. 19, 2014, entitled “IMPROVEDMETHODS OF CELL CULTURE FOR ADOPTIVE CELL THERAPY,” (hereinafter the“parent case”) which claims the benefit of U.S. Provisional ApplicationNo. 61/267,761, filed Dec. 8, 2009, also entitled “IMPROVED METHODS OFCELL CULTURE FOR ADOPTIVE CELL THERAPY”, which are herein incorporatedby reference in their entirety.

FIELD OF THE INVENTION

The present invention relates generally to methods of culturing cells,and more specifically to culturing cells for cell therapy.

BACKGROUND

Cell culture is major contributor to the cost and complexity of celltherapy. With current methods, the process of culturing the cells istime consuming and expensive. Typically, to produce a large number ofcells, an in vitro culture process is undertaken that proceeds instages. At the earliest stage, the desired cells are a relatively smallpopulation within a composition of cells that are placed into cellculture devices. In this stage, the composition of cells typicallyincludes the source of the desired cells (such as peripheral bloodmononuclear cells), feeder cells that stimulate growth of the desiredcells, and/or antigen presenting. Culture devices and methods that allowthe medium that cells reside in to be in a generally undisturbed stateare favored since the cells remain relatively undisturbed. Such devicesinclude standard tissue culture plates, flasks, and bags. The cultureprogresses in stages generally consisting of allowing the cellcomposition to deplete the medium of growth substrates such as glucose,removing the spent medium, replacing the spent medium with fresh medium,and repeating the process until the desired quantity of desired cells isobtained. Often, the cell composition is moved to other devices toinitiate a new stage of production as the desired cell populationincreases and additional growth surface is needed. However, withconventional methods, the rate of population growth of the desired cellsslows as the population of cells upon the growth surface increases. Theend result is that it is very time consuming and complicated to producea sizable population of desired cells.

State of the art production methods for generating T lymphocytes withantigen specificity to Epstein Barr virus (EBV-CTLs) provide an exampleof production complexity. The conventional method for optimal expansionof EBV-CTLs uses standard 24-well tissue culture plates, each wellhaving 2 cm² of surface area for cells to reside upon and the mediumvolume restricted to 1 ml/cm² due to gas transfer requirements. Theculture process begins by placing a cell composition comprised of PBMC(peripheral blood mononuclear cells) in the presence of an irradiatedantigen presenting cell line, which may be a lymphoblastoid cell line(LCL), at a surface density (i.e. cells/cm² of growth surface) ratio ofabout 40:1 with about 1×10⁶ PBMC/cm² and 2.5×10⁴ irradiated antigenpresenting cells/cm². That instigates the population of EBV-CTLs withinthe cell composition to expand in quantity. After 9 days, EBV-CTLs areselectively expanded again in the presence of irradiated antigenpresenting LCL at a new surface density ratio of 4:1, with a minimumsurface density of about 2.5×10⁵ EBV-CTL/cm². Medium volume is limitedto a maximum ratio of 1 ml/cm² of growth surface area to allow oxygen toreach the cells, which limits growth solutes such as glucose. As aresult, the maximum surface density that can be achieved is about 2×10⁶EBV-CTL/cm². Thus, the maximum weekly cell expansion is about 8-fold(i.e. 2×10⁶ EBV-CTL/cm² divided by 2.5×10⁵ EBV-CTL/cm²) or less.Continued expansion of EBV-CTLs requires weekly transfer of the EBV-CTLsto additional 24-well plates with antigenic re-stimulation, and twiceweekly exchanges of medium and growth factors within each well of the24-well plate. Because conventional methods cause the rate of EBV-CTLpopulation expansion to slow as EBV-CTL surface density approaches themaximum amount possible per well, these manipulations must be repeatedover a long production period, often as long as 4-8 weeks, to obtain asufficient quantity of EBV-CTLs for cell infusions and quality controlmeasures such as sterility, identity, and potency assays.

The culture of EBV-CTLs is but one example of the complex cellproduction processes inherent to cell therapy. A more practical way ofculturing cells for cell therapy that can reduce production time andsimultaneously reduce production cost and complexity is needed.

We have created novel methods that increase the population growth ratethroughout production, and by so doing, reduce the complexity and timeneeded to produce cells.

Primary non-adherent cells such as antigen specific T cells, naturalkiller cells (NK), regulatory T cells (Treg), tumor infiltratinglymphocytes (TIL), marrow infiltrating lymphocytes (TIL), and islets areoften the focus of production. Many production processes aim to increasethe population of desired cells, often referred to as effector cells,often in co-culture conditions that rely on other cell types tostimulate growth and/or antigen specificity of the desired cells. Thecells used in co-culture are often referred to as feeder cells and/orantigen presenting cells. In some cases, co-cultures transition toexpansion of the desired cell population in the absence of feeder and/orantigen presenting cells such as TIL production. Production of antigenpresenting cells and/or feeder cells in the absence of effector cells isalso prevalent. Also, sometimes culture is intended to maintain healthof a cell population as opposed to increasing the population per se,such as islet culture for treatment of diabetes. Thus, culture devicesand production processes for cell culture in Adoptive Cell Therapy mustdeal with many possible production applications.

For Adoptive Cell Therapy to be useful on a wide scale, the cellproduction process needs to be greatly simplified and made lessexpensive. However, state of the art devices and methods for productionare not capable of making that happen. A brief explanation of why thatis the case follows.

Devices currently relied upon extensively in the field of Adoptive CellTherapy are static cell culture devices, namely cell culture plates,flasks, and gas permeable bags. These static devices are intended toallow cells to reside in proximity of one another during culture inorder to facilitate communication between co-cultures and/or allow nonco-cultures to remain physically quiescent. The physically undisturbedstate is beneficial for a variety of biological reasons as skilledartisans are well aware. Furthermore, static cell culture devices areuncomplicated and do not require constant use of ancillary equipmentduring their operation to perfuse medium or gas through the device,agitate the medium such as by sparging, stirring or shaking theapparatus, and/or keep cells from settling to the bottom of the device.Thus, static devices are compatible with standard laboratory and cellculture equipment such as incubators, and have minimal or no reliance onancillary equipment. Although static devices have the describedadvantages, they also have inherent problems that prevent efficient andpractical production of cells for Adoptive Cell Therapy.

Among the inherent problems are the limited height at which medium canreside above the growth surface, ranging from an upper limit of about0.3 cm in plates and flasks, according to manufacturer'srecommendations, to 2.0 cm in gas permeable bags. Thus, plates andflasks have a limited medium volume to growth surface area ratio of nomore than 0.3 ml/cm² and gas permeable bags are constrained to no morethan 2.0 ml/cm². Compounding the design limits of plates, flasks, andbags are the state of the art protocols for their use in the field ofAdoptive Cell Therapy, which narrowly constrain cell density to therange of 0.5 to 2.0×10⁶ cells/ml and which inherently rely on a surfacedensity of at least 0.5×10⁶ cells/cm² to initiate culture. These limitslead to a variety of problems that render cell production for AdoptiveCell Therapy impractical, including an excessive amount of devices inthe process, an inordinate amount of labor to maintain cultures, a highrisk of contamination, and/or long duration of time to produce cells.Bags have unique problems in that routine handling of the bag causescells to be disturbed from their resting location and distributed intothe media.

Alternative devices to the plate, flask, and bag have been introduced inco-pending U.S. Publication Nos. 2005/0106717 A1 to Wilson et al.(hereinafter referred to as Wilson '717) and 2008/0227176 A1 to Wilson(hereinafter referred to as Wilson '176), and alternative methods forculture have been introduced in the parent case which discloses aparticularly powerful improvement of cell production process for thefield of Adoptive Cell Therapy. Wilson '717 describes various innovativegas permeable devices that allow culture methods to be performed byscale up in the vertical direction, moving beyond the limited mediumheight and limited medium volume to growth surface area ratios ofplates, flasks, and bags to allow more efficient use of physical space.Wilson '176 builds upon Wilson '717 by allowing even more growth area toreside in a given amount of physical space. The parent case disclosesdiscoveries that allow more efficient co-culture of cells commonly usedin the field of Adoptive Cell Therapy, including teaching away fromstate of the art limits relating to cell surface density in order toprovide a wide range of unexpected benefits.

The present invention builds upon the parent case with new discoveriesthat further improve the efficiency and practicality of cell production,particularly for the field of Adoptive Cell Therapy, and builds uponWilson '717 and Wilson '176 to enable various novel methods disclosedherein.

SUMMARY

It has been discovered that the production of cells for cell therapy canoccur in a shorter time period and in a more economical manner than iscurrently possible by using a staged production process that allowsunconventional conditions to periodically be re-established throughoutthe production process. The unconventional conditions include reducedsurface density (i.e. cells/cm²) of desired cells, novel ratios ofdesired cells to antigen presenting and/or feeder cells, and/or use ofgrowth surfaces comprised of gas permeable material with increasedmedium volume to surface area ratios.

Embodiments of this invention relate to improved methods of culturingcells for cell therapy applications. They include methods that reducethe time, cost, and complexity needed to generate a desired number ofdesired cells by use of various novel methods that allow the desiredcell population to maintain a higher growth rate throughout theproduction process relative to conventional methods.

One aspect of the present invention relies on conducting the cultureprocess in stages and establishing conditions at the onset of one ormore stages that allow the growth rate of the desired cell population toexceed what is currently possible. At least one stage of culture, andpreferably nearly all, establish initial conditions that include thedesired cells resting either on non-gas permeable or gas permeablegrowth surfaces at unconventionally low surface density and at anunconventional ratio of antigen presenting cells (and/or feeder cells)per desired cell. By using the novel embodiments of this aspect of theinvention, the desired cell population can experience more doublings ina shorter period of time than allowed by conventional methods, therebyreducing the duration of production.

Another aspect of the present invention relies on conducting the cultureprocess in stages and establishing conditions at the onset of one ormore stages such that the growth rate of the desired cell populationexceeds what is currently possible. At least one stage of culture, andpreferably nearly all, establish conditions that include the desiredcells resting on a growth surface comprised of gas permeable material atunconventionally high medium volume to growth surface area ratios. Byusing the novel embodiments of this aspect of the invention, the desiredcell population can experience more doublings in a shorter period oftime than is allowed by conventional methods, thereby reducing theduration of production.

Another aspect of the present invention relies on conducting the cultureprocess in stages and establishing conditions of each stage such thatthe growth rate of the desired cell population exceeds what is currentlypossible. At least one stage of culture, and preferably nearly all,establish initial conditions that include the desired cells resting ongrowth surfaces comprised of gas permeable material at unconventionallylow surface density (i.e. cells/cm²) with an unconventional ratio ofantigen presenting cells (and/or feeder cells) per desired cell and inthe presence of unconventionally high medium volume to growth surfacearea ratios. By using the novel embodiments of this aspect of theinvention, the desired cell population can experience more doublings ina shorter period of time than conventional methods allow, therebyreducing the duration of production.

We have discovered additional methods of cell culture that teach awayfrom state of the art methods in the field of Adoptive Cell Therapy andbuild upon the disclosures of the parent case to make the process ofculturing and/or preparing cells more practical and cost effective thancurrent methodologies.

In one embodiment of the present invention using gas permeable cellculture devices to culture cells, cells are capable of initiatingoutgrowth when residing in a gas permeable device from a state whereinsurface density (cells/cm²) and cell density (cells/ml) are reducedbelow conventional methods.

In another embodiment of the present invention using gas permeable cellculture devices to culture cells, the need to count cells to determinehow many cells are in culture at any given time can be replaced bytaking a sample of solutes in the medium and using it to predict thepopulation within the culture at any given time.

In another embodiment of the present invention using gas permeable cellculture devices to culture cells, medium volume to growth surface areais increased in order to reduce the frequency of feeding relative tostate of the art methods or even eliminate the need to feed the culturealtogether after culture onset.

In another embodiment of the present invention using gas permeable cellculture devices to culture cells, medium volume to growth surface areais further increased in order to allow a longer period of time at whicha cell population can reside at high viability after reaching itsmaximum population.

In another embodiment of the present invention, gas permeable cellculture and cell recovery devices are disclosed that are capable ofreducing the medium volume in a culture without cell loss, concentratingcells absent the need for centrifugation, and increasing cell densityprior to removing cells from the devices.

In another embodiment of the present invention, methods of use for novelgas permeable cell culture and cell recovery devices are disclosed thatare capable of reducing the medium volume in a culture without cell lossin order to minimize need to increase the number of devices in cultureshould an operator choose to feed the culture.

In another embodiment of the present invention using gas permeable cellculture devices to culture cells, methods of rapidly producing CAR Tcells and improving killing capacity by use of APCs in culture aredisclosed.

In another embodiment of the present invention using gas permeable cellculture devices to culture cells, methods of the present invention arelinearly scalable in direct proportion to increase in the surface areaof the growth surface.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention may be more completely understood in consideration of thefollowing detailed description of various embodiments of the inventionin connection with the accompanying drawings, in which:

FIG. 1A shows the population of antigen-specific T-cells in Example 1undergoes at least 7 cell doublings after the initial stimulation overthe first 7 days.

FIG. 1B shows data demonstrating the magnitude of expansion of a T-cellpopulation within a cell composition over time as determined by tetrameranalysis for Example 1.

FIG. 1C the rate of population growth of antigen-specific T-cellsdiminishes over a 23 day period in Example 1.

FIG. 2 shows a table that illustrates the discrepancy between thepotential expansion and observed fold expansion of antigen-specificT-cells in Example 1.

FIG. 3A shows the presence of antigen-specific T-cells followingstimulations in Example 2.

FIG. 3B shows the expansion of a population of antigen-specific T-cellsas surface densities diminish from 1×10⁶/cm² to 3.1×10⁴/cm² whilemaintaining an antigen-specific T-cell to antigen presenting cell ratioof 4:1 in Example 2.

FIG. 3C shows the expansion of a population of antigen-specific T-cellsas surface densities diminish from 1×10⁶/cm² to 3.1×10⁴/cm² while in thepresence of a fixed number of antigen presenting cells in Example 2.

FIG. 4 shows an example of results obtained when continuing the workdescribed in FIG. 3, which further demonstrated that when desired cellsneed the support of other cells, unconventionally low desired cellsurface density can initiate population expansion so long as desiredcells are in the presence of an adequate supply of feeder and/or antigenpresenting cells.

FIG. 5 shows a histogram demonstrating the ability to repeat themagnitude of the population expansion of desired cells by initiatingculture at three differing cell surface densities (CTL/cm²).

FIG. 6 shows a cross-sectional view of a gas permeable test fixture usedto generate data.

FIG. 7A shows the growth curves of antigen-specific T cells produced inaccordance with the present invention in comparison to conventionalmethods as undertaken in Example 5.

FIG. 7B shows that for Example 5, cell viability was significantlyhigher in antigen-specific T cells produced in accordance with thepresent invention in comparison to conventional methods as determined byflow cytometric forward vs. side scatter analysis.

FIG. 7C shows that for Example 5, cell viability was significantlyhigher in antigen-specific t cells produced in accordance with thepresent invention in comparison to conventional methods as determined byAnnexin-PI 7AAD.

FIG. 7D showed that for Example 5, the superior growth of cells producedin the novel methods of the present invention exhibited the same cellspecific growth rate as cell cultured using conventional methods asdetermined by daily flow cytometric analysis of CFSE labeled cells,confirming that the increased rate of cell expansion resulted fromdecreased cell death.

FIG. 8A shows how EVB-CTLs were able to expand beyond what was possiblein conventional methods without need to exchange medium.

FIG. 8B shows how the culture condition of Example 6 did not modify thefinal cell product as evaluated by Q-PCR for EBER.

FIG. 8C shows how the culture condition of Example 6 did not modify thefinal cell product as evaluated by Q-PCR for B cell marker CD20.

FIG. 9 shows an illustrative example in which we experimentallydemonstrated that a very low cumulative surface density of desired cellsand antigen presenting cells (in this case AL-CTLs and LCLs cellscombining to create a cell composition with a surface density of 30,000cells/cm²) was unable to initiate outgrowth of the AL-CTL population.

FIG. 10A presents data of Example 8 that show how two novel methods ofculturing cells produce more cells over a 23 day period than aconventional method.

FIG. 10B shows a photograph of cells cultured in a test fixture inExample 8.

FIG. 10C shows that in Example 8, the two novel methods of culture andthe conventional method all produce cells with the same phenotype.

FIG. 10D shows that for Example 8, a representative culture in whichT-cells stimulated with EBV peptide epitopes from LMP1, LMP2, BZLF1 andEBNA1 of EBV and stained with HLA-A2-LMP2 peptide pentamers stainingshowed similar frequencies of peptide-specific T-cells.

FIG. 10E shows that for the novel methods and the conventional method ofExample 8, cells maintained their cytolytic activity and specificity andkilled autologous EBV-LCL, with low killing of the HLA mismatchedEBV-LCL as evaluated by ⁵¹Cr release assays.

FIG. 11 shows a graphical representation of expansion of a desired cellpopulation on a growth surface under the conventional scenario ascompared to population expansion of the desired cell type using oneaspect of the present invention.

FIG. 12 shows an example of the advantages that can be obtained byutilizing a growth surface comprised of gas permeable material and anunconventionally high medium volume to growth surface area ratio beyond1 or 2 ml/cm².

FIG. 13 shows a graphical representation of a novel method of expansionof a desired cell population on a growth surface under the conventionalscenario as compared to population expansion of the desired cell typeunder one embodiment of the present invention in which the cell surfacedensity at the completion of is much greater than conventional surfacedensity.

FIG. 14 shows another novel method of cell production that provides yetfurther advantages over conventional methods.

FIG. 15 shows a comparison of each production method depicted in FIG. 14to demonstrate the power of the novel method and why it is useful toadjust the production protocol at various stages to fully capture theefficiency.

FIG. 16 shows an example of how one could adjust the production protocolin the novel method to gain efficiency as production progresses.

FIG. 17A shows a representative spreadsheet of the experimentalconditions at 1.0E+06 cells/cm² and results.

FIG. 17B shows a representative spreadsheet of the experimentalconditions at 0.5E+06 cells/cm² and results.

FIG. 17C shows a representative spreadsheet of the experimentalconditions at 0.25E+06 cells/cm² and results.

FIG. 17D shows a representative spreadsheet of the experimentalconditions at 0.125E+06 cells/cm² and results.

FIG. 17E shows a representative spreadsheet of the experimentalconditions at 0.0625E+06 cells/cm² and results.

FIG. 18 compares the fold expansion of the population increase relativeto the surface density of each of the experimental conditions detailedin FIG. 17A-FIG. 17E.

FIG. 19A shows a representative spreadsheet of the experimentalconditions and typical results for the culture of K562 cells underequivalent starting conditions except for the glucose concentration.

FIG. 19B shows cell population expansion under two starting glucosecondition over a time period of 11 days.

FIG. 19C shows the glucose depletion rate in each culture condition.

FIG. 19D shows the glucose consumption rate in each culture condition.

FIG. 19E shows an overlay of the predicted number of cells in apopulation using the formulaic calculation, versus the number of cellsas determined by manual counts for the culture initiated at a glucoseconcentration of 240 mg/dl.

FIG. 19F shows an overlay of the predicted number of cells in apopulation using the formulaic calculation, versus the number of cellsas determined by manual counts for the culture initiated at a glucoseconcentration of 240 mg/dl.

FIG. 20 shows a graphical representation of population growth,normalized for growth surface area, under various medium feedingconditions.

FIG. 21 shows a spreadsheet that summarizes conditions on day 0, day 9,and day 16 for an experiment that demonstrated the capability of usingglucose depletion as a surrogate measure of cell population.

FIG. 22A shows a cross-sectional view of one example of an embodiment ofa present invention of a cell culture and cell recovery device 1000configured to perform the disclosed novel cell culture and/or novel cellrecovery methods.

FIG. 22B shows cell culture and cell recovery device 1000 in an initialstate of static culture at the onset of any given cell production stageof culture.

FIG. 22C shows cell culture and cell recovery device 1000 prepared torecover cells in a reduced volume of medium.

FIG. 22D shows the process of reorienting cell culture and cell recoverydevice 1000 into a position at an angle 1026 that deviates from theoriginal horizontal cell culture position in order to relocate cellrecovery medium 1024.

FIG. 23A shows the conditions of Evaluation A at the onset of cultureand as the culture progressed.

FIG. 23B shows the conditions of Evaluation B at the onset of cultureand as the culture progressed.

FIG. 23C shows the conditions of Evaluation C at the onset of cultureand as the culture progressed.

FIG. 23D shows the total live cells in culture at various time points inthe culture.

FIGS. 23E1-E3 show the percentage of CAR T cell expression at the onsetof culture and at the completion of culture.

FIG. 23F shows the total fold expansion of CAR T cells during culture.

FIG. 23G demonstrates the prediction of the live cell population inEvaluation A was representative of cell population as determined bymanual counts.

FIG. 23H shows the capacity of cells obtained from Condition A andCondition B to kill tumor cells expressing PSCA.

FIG. 24A is a side by side comparison of the population expansion ofCART cells specific to PSCA.

FIG. 24B is a side by side comparison of the population expansion of CART cells specific to Muc1.

FIG. 24C is a graph of population expansion of CART cells.

FIG. 24D is a graph of population expansion of Muc1 cells.

FIG. 24E shows the population growth curves of three gas permeableculture devices with differing growth areas.

FIG. 24F shows the population growth of FIG. 24E curves after beingnormalized to surface density.

DETAILED DESCRIPTION Definitions

-   Adherent cells: Cell that attach to growth surface-   Antigen presenting cells (APC): Cells that act to trigger the    desired cells to respond to a particular antigen.-   CTL: Cytotoxic T cell-   Cell density: The ratio of cells number per unit volume of medium    (cells/ml)-   Desired cells: The specific type of cell that that the production    process aims to expand and/or recover in quantity. Generally the    desired cells are non-adherent and examples include regulatory T    cells (Treg), natural killer cells (NK), tumor infiltrating    lymphocytes (TIL), primary T lymphocytes and a wide variety of    antigen specific cells, and many others (all of which can also be    genetically modified to improve their function, in-vivo persistence    or safety). Cells required for clinical use can be expanded with    feeder cells and/or antigen presenting cells that can include PBMC,    PHA blast, OKT3 T, B blast, LCLs and K562, (natural or genetically    modified to express and antigen and/or epitope as well as    co-stimulatory molecules such as 41BBL, OX40, CD80, CD86, HLA, and    many others) which may or may not be pulsed with peptide or other    relevant antigens.-   EBV: Epstein Barr Virus-   EBV-CTL: A T-cell that specifically recognized EBV-infected cells or    cells expressing or presenting EBV-derived peptides through its T    cell surface receptor.-   EBV-LCL: Epstein Barr virus transformed B lymphoblastoid cell line.-   Feeder cells: Cells that act to cause the desired cells to expand in    quantity. Antigen presenting cells can also act as feeder cells in    some circumstances.-   Growth surface: The area within a culture device upon which cells    rest.-   Initiating culture: Generally refers to the conditions at the onset    of a culture process and/or at the onset of production cycles-   Medium exchange: Synonymous with feeding the cells and is generally    the process by which old medium is replenished with fresh medium-   PBMCs: Peripheral Blood Mononuclear Cells derived from peripheral    blood, which are a source of some of the desired cells and which can    act as feeder cells.-   Responder (R): A cell that will react to a stimulator cell.-   Static cell culture: A method of culturing cells in medium that is    not stirred or mixed except for occasions when the culture device is    moved from location to location for routine handling and/or when    cells are periodically fed with fresh medium and the like. In    general, medium in static culture is typically in a quiescent state.    It is not subjected to forced movement such as occurs in perfusion    systems (in which medium is constantly moved through the vessel),    shaker systems in which the culture device is physically shaken to    move the medium, stirred systems (in which a stir bar moves within    the device to agitate medium and cells), or any other mechanisms or    equipment used to force medium to be moved and mixed throughout the    duration of culture. Cells gravitate to growth surfaces in the    devices where they reside in an undisturbed state except for periods    of occasional feeding, at which point the culture is provide with    fresh medium by first removing medium and then adding medium, by    adding medium without removing medium, or by removing medium and    cells and distributing the medium and cells to new devices and    adding fresh medium to those devices. Pumps to aid the feeding    process are not uncommon. For example gas permeable cell culture    bags often rely on gravity or pumps to move fluid to and from them    in a closed system manner. The vast majority of the culture duration    is one in which cells and medium reside in a quiescent and    un-agitated state. This invention is directed to static cell culture    methods.-   Stimulated: The effect that antigen presenting and/or feeder cells    have on the desired cells.-   Stimulator (S): A cell that will influence a responder cell.-   Surface density: The quantity of cells per unit area of the growth    surface within the device upon which the cells rest.-   Suspension cells: Cell that do not need to attach to growth surface,    synonymous with non-adherent cells

In attempting to find novel methods to simplify the production of adesired population of cells for adoptive T cell therapy, a series ofexperiments were conducted that have that opened the door to moreefficient culture of cells for cell therapy applications. Numerousillustrative examples and various aspects of the present invention aredescribed to indicate how the ability to reduce production time andcomplexity relative to conventional methods can be achieved.

EXAMPLE 1

Demonstration of limitations of conventional methods.

The data of this example demonstrate the limits of conventional culturemethods for the production of EBV-CTL in standard 24 well tissue cultureplates (i.e. 2 cm² surface area per well) using a medium volume of 2 mlper well (i.e. medium height at 1.0 cm and a medium volume to surfacearea ratio of 1 ml/cm²).

Stage 1 of culture, day 0: The expansion of an EBV-CTL population wasinitiated by culturing a cell composition of PBMCs from normal donors(about 1×10⁶ cells/ml) with antigen presenting gamma-irradiated (40 Gy)autologous EBV-LCLs at a 40:1 ratio (PBMC:LCLs) and a medium volume togrowth surface ratio of 1 ml/cm² thereby establishing a cell compositionsurface density of about 1×10⁶ cells/cm² in RPMI 1640 supplemented with45% Click medium (Irvine Scientific, Santa Ana, Calif.), with 2 mMGlutaMAX-I, and 10% FBS.

Stage 2 of culture, day 9-16: On day 9, EBV-CTLs were harvested from thecell composition created in Stage 1, resuspended in fresh medium at asurface density of 0.5×10⁶ EBV-CTL/cm² and re-stimulated with irradiatedautologous EBV-LCLs at a ratio 4:1 CTL:LCL (surface density 0.5×10⁶CTL/cm²:1.25×10⁵ LCL/cm²). On day 13, 1 ml of the 2 ml medium volume ineach well of the 24-well plates was removed and replaced with 1 ml offresh medium containing recombinant human IL-2 (IL-2) (50 U/mL)(Proleukin; Chiron, Emeryville, Calif.)

Stage 3 of culture, day 17-23: The conditions of Stage 2 were repeatedwith twice weekly addition of IL-2 and the culture was terminated on day23. Although the culture was terminated, it could have been continuedwith additional culture stages that mimicked that of stages 2 and 3.

Cell lines and tumor cells for use as target cells in Cytotoxicityassays: BJAB (a B cell lymphoma) and K562 (a chronic erythroid leukemia)were obtained from the American Type Culture Collection (ATCC,Rockville, Md., USA). All cells were maintained in culture with RPMI1640 medium (GIBCO-BRL, Gaithersburg, Md.) containing 10%heat-inactivated fetal calf serum (FCS), 2 mM L-glutamine, 25 IU/mLpenicillin, and 25 mg/mL streptomycin (all from BioWhittaker,Walkersville, Md.). Cells were maintained in a humidified atmospherecontaining 5% CO₂ at 37° C.

Immunophenotyping:

Cell surface: Cells were stained with Phycoerythrin (PE), fluoresceinisothiocyanate (FITC), periodin chlorophyll protein (PerCP) andallophycocyanin (APC)-conjugated monoclonal antibodies (MAbs) to CD3,CD4, CD8, CD56, CD16, CD62L, CD45RO, CD45RA, CD27, CD28, CD25, CD44 fromBecton-Dickinson (Mountain View, Calif., USA). PE-conjugated tetramers(Baylor College of Medicine) and APC-conjugated pentamers (ProimmuneLtd, Oxford, UK), were used to quantify EBV-CTL precursor frequencies.For cell surface and pentamer staining 10,000 and 100,000 live events,respectively, were acquired on a FACSCalibur flow cytometer and the dataanalyzed using Cell Quest software (Becton Dickinson).

CFSE labeling to measure cell division: To assess the doubling rate of2×10⁷ PBMC or EBV-specific CTLs (EBV-CTLs) were washed twice andresuspended in 850 μl 1× phosphate-buffered saline (PBS) containing 0.1%Fetal Bovine Serum (FBS) (Sigma-Aldrich). Prior to staining, an aliquotof carboxy-fluorescein diacetate, succinimidyl ester (CFSE) (10 mM indimethyl sulfoxide) (Celltrace™ CFSE cell proliferation kit (C34554)Invitrogen) was thawed, diluted 1:1000 with 1× PBS and 150 μl of thedilution was added to the cell suspension (labeling concentration was 1μM). Cells were incubated with CFSE for 10 minutes at room temperature.Subsequently 1 ml FBS was added to the cell suspension followed by a 10minute incubation at 37° C. Afterwards cells were washed twice with 1×PBS, counted, and stimulated with antigen as described.

AnnexinV-7-AAD staining: To determine the percentage of apoptotic andnecrotic cells in our cultures we performed Annexin-7-AAD staining asper manufacturers' instructions (BD Pharmingen™ #559763, San Diego,Calif.). Briefly, EBV-CTL from the 24-well plates or the G-Rex werewashed with cold PBS, resuspended in 1× Binding Buffer at aconcentration of 1×10⁶ cells/ml, stained with Annexin V-PE and 7-AAD for15 minutes at RT (25° C.) in the dark. Following the incubation thecells were analyzed immediately by flow cytometry.

Chromium release assay: We evaluated the cytotoxic activity of EBV-CTLsin standard 4-hour ⁵¹Cr release assay, as previously described. Asdesired cells we used autologous and HLA class I and II mismatchedEBV-transformed lymphoblastoid cell line (EBV-LCL) to measure MHCrestricted and unrestricted killing, as well as the K562 cell line tomeasure natural killer activity. Chromium-labeled desired cellsincubated in medium alone or in 1% Triton X-100 were used to determinespontaneous and maximum ⁵¹Cr release, respectively. The mean percentageof specific lysis of triplicate wells was calculated as follows: [(testcounts . . . spontaneous counts)/(maximum counts spontaneouscounts)]×100.

Enzyme-Linked Immunospot (ELIspot) assay: ELIspot analysis was used toquantify the frequency and function of T cells that secreted IFNγ inresponse antigen stimulation. CTL lines expanded in 24 well plates or inthe G-Rex were stimulated with irradiated LCL (40Gy) or LMP1, LMP2,BZLF1 and EBNA1 pepmixes (diluted to 1 μg/ml) (JPT Technologies GmbH,Berlin, Germany), or EBV peptides HLA-A2 GLCTLVAML=GLC, HLA-A2CLGGLLTMV=CLG, HLA-A2-FLYALALLL=FLY, and HLA-A29 ILLARLFLY=ILL (GenemedSynthesis, Inc. San Antonio, Tex.), diluted to a final concentration of2 μM, and CTLs alone served as a negative controls. CTLs wereresuspended at 1×10⁶/ml in ELIspot medium [(RPMI 1640 (Hyclone, Logan,Utah) supplemented with 5% Human Serum (Valley Biomedical, Inc.,Winchester, Va.) and 2-mM L-glutamine (GlutaMAX-I, Invitrogen, Carlsbad,Calif.)].

Ninety-six-well filtration plates (MultiScreen, #MAHAS4510, Millipore,Bedford, Mass.) were coated with 10 μg/mL anti-IFN-γ antibody(Catcher-mAB91-DIK, Mabtech, Cincinnati, Ohio) overnight at 4° C., thenwashed and blocked with ELIspot medium for 1 hour at 37° C. Responderand stimulator cells were incubated on the plates for 20 hours, then theplates were washed and incubated with the secondary biotin conjugatedanti-IFN-γ monoclonal antibody (Detector-mAB (7-B6-1-Biotin), Mabtech)followed by incubation with Avidin:biotinylated horseradish peroxidasecomplex (Vectastain Elite ABC Kit (Standard), #PK6100, VectorLaboratories, Burlingame, Calif.) and then developed with AEC substrate(Sigma, St. Louis, Mo.). Each culture condition was run in triplicate.Plates were sent for evaluation to Zellnet Consulting, New York, N.Y.Spot-forming units (SFC) and input cell numbers were plotted.

Statistical analysis: In vitro data are presented as mean±1 SD.Student's t test was used to determine the statistical significance ofdifferences between samples, and P<0.05 was accepted as indicating asignificant difference.

Under these culture conditions, the population of antigen-specificT-cells undergoes at least 7 cell doublings after the initialstimulation over the first 7 days, as shown in FIG. 1A. Thus we expect aweekly T-cell expansion of 128-fold (as measured by the frequency ofantigen-specific T-cells times the total number of cells in the cellcomposition). The frequency of tetramer positive cells after the first,second, and third stimulations is shown in FIG. 1B. On day 0 thefrequency of T-cells reactive against two EBV tetramers, RAK and QAK was0.02% and 0.01%, respectively. After a single stimulation on day 0, byday 9 the frequency of tetramer-positive T-cells in the cell compositionhad increased from 0.02% and 0.01% to 2.7% and 1.25%, respectively.Thus, a 135-fold and 125-fold increase in the percentage ofantigen-specific tetramer positive T-cells residing within the cellcomposition was attained as measured by RAK and QAK. Also, after asingle stimulation on stage 1 of culture, day 0, a 1.1 fold increase inthe surface density of cells in the cell composition (data not shown)was observed by day 9 (approximately 1.1×10⁶ cells/cm² were present).Since the majority of cells within the PBMC composition are not specificfor the stimulating antigens, little overall increase in total cellnumber is observed, but the fold expansion of the antigen-specific cellpopulation within the composition was around 280 during the first stageof culture, as shown in FIG. 1C. Unfortunately, although the number ofcell doublings was the same during the second and third stages ofculture as measured by CSFE, this rate of antigen-specific T cellexpansion was not sustained during the 2^(nd) or the 3^(rd) stages ofculture, being only 5.7 in stage two and 4.3 in stage three. FIG. 2shows a table that illustrates the discrepancy between the potentialexpansion and observed fold expansion of antigen-specific T-cells (n=3).

Example 1 demonstrates that the amount of time it takes to produce thedesired cells is typically delayed after roughly the first week ofproduction since the rate of population expansion of the desired cellsdecreases in subsequent stages of culture.

EXAMPLE 2

Reducing the amount of time needed to increase the desired cellpopulation can be achieved by reducing the cell surface density of thedesired cell population as the onset of any given stage or stages ofculture.

We hypothesized that the decreased rate of expansion of the desired cellpopulation following the second T-cell stimulation compared to the firststimulation was due to limiting cell culture conditions that resulted inactivation induced cell death (AICD). For example, referring to FIG. 3A,at the first stimulation, the EBV antigen-specific T-cell component ofPBMCs represents, at most, 2% of the population and so theantigen-specific responder T-cell seeding density is less than 2×10⁴ percm², with the remaining PBMC acting as non-proliferating feeder cells(seen as the CFSE positive cells in FIG. 3A) that sustain optimalcell-to-cell contact allowing proliferation of the antigen-specificCTLs. By contrast, at the second stimulation on day 9, the majority ofT-cells are antigen-specific, and although the total cell density of thecomposition is about the same, the proliferating cell density is 50 to100 fold higher. As a consequence, on re-stimulation the majority ofcells proliferate and may therefore rapidly consume and exhaust theirnutrients and O₂ supply.

To determine whether limiting culture conditions were responsible forsub-optimal T cell growth rates, we measured the expansion of activatedT-cells plated at lower cell densities. Methods were as previouslydescribed in Example 1.

We seeded activated EBV-specific T-cells in wells of standard 24-wellplates, each well having 2 cm² of growth surface area, at doublingdilutions to create diminishing surface densities ranging from 1×10⁶/cm²to 3.1×10⁴/cm² while maintaining a responder cell to stimulatory cellratio (R:S) of 4:1 as shown in FIG. 3B. The maximum CTL expansion(4.7±1.1 fold) was achieved with a starting CTL surface density of1.25×10⁵ per cm², but further dilution decreased the rate of expansionas shown in FIG. 3B. We speculated that this limiting dilution effectwas possibly due to lack of cell-to-cell contact, and therefore wecultured doubling dilutions of EBV-CTL from surface densities of 1×10⁶to 3.1×10⁴ with a fixed number of feeder cells (EBV-LCL plated at asurface density of 1.25×10⁵/cm²) and assessed cell expansion over a 7day period. We observed a dramatic increase in CTL expansion from merely2.9±0.8 fold with EBV-CTL at a surface density of 1×10⁶/cm² all the wayto a 34.7±11 fold expansion with EBV-CTL at a surface density of3.1×10⁴/cm², as presented in FIG. 3C. Importantly, this modification ofthe culture conditions did not change the function or antigenspecificity of the cells (data not shown). A population of activatedantigen-specific T cells is therefore capable of greater expansion thanconventional culture methods allow. Of note, the maximum surface densityachieved after stimulation (1.7 to 2.5×10⁶/cm²) was the same regardlessof the starting surface density.

Thus, conventional culture conditions were limiting, indicating themedium volume to growth surface area ratio needs to increase beyond theconventional 1 ml/cm² to allow the desired cell population to movebeyond the surface density limits of conventional methods. Additionally,improved expansion of antigen-specific CTL to about 34-fold can beobtained by reducing the surface density of the desired cell populationbelow conventional methods at the onset of any stage of culture. Thishas substantial ramifications in cell therapy, where the quantity ofcells at the onset of production is often quite limited. For example, bydistributing the in limited amount of desired cells onto increasedsurface area at lowered surface density, a greater population of desiredcells can be attained in a shorter period of time as the rate ofpopulation growth increases dramatically relative to conventionalsurface density.

EXAMPLE 3

A minimum surface density of a cell population that includes the desiredcells and/or antigen presenting cells can allow outgrowth of a desiredcell population that is seeded at very low surface density.

FIG. 4 shows an example of results we obtained when continuing the workdescribed in FIG. 3, which further demonstrated that when desired cellsneed the support of other cells, unconventionally low desired cellsurface density can initiate population expansion so long as desiredcells are in the presence of an adequate supply of feeder and/or antigenpresenting cells. In these experiments, we continue to demonstrate how atotal cell composition with a surface density and R:S ratio of betweenabout 1.0×10⁶ desired cells/cm² at an R:S ratio of 8 to 1 and merelyabout 3900 desired cells/cm² at an R:S ratio of 1 to 32 could allowdesired cells to be greatly expanded to over 50 fold times the startingsurface density, at which point we discontinued testing.

EXAMPLE 4

The ability to allow a production process to repeat in stages byinitiating a stage with an unconventionally low desired cell surfacedensity, allowing population expansion, terminating the stage andrepeating conditions was demonstrated to deliver repeatable outcomes.

We continued the assessments described in Example 3 at three of thedesired cell surface densities (CTL/cm²) as shown in FIG. 5. Eachspecific seeding density was able to consistently attain the same foldexpansion. The implications will be described in more detail further onas they relate to the ability to dramatically reduce the production timefor a desired cell population.

EXAMPLE 5

Culturing desired cells on a growth surface that is comprised of gaspermeable material while simultaneously increasing the medium volume togrowth surface area ratio increases the number of times a desired cellpopulation can double in a given stage of culture relative toconventional methods and increases the surface density that isattainable.

Cell lines and tumor cells, immunophenotyping, CFSE labeling,AnnexinV-7-AAD staining, chromium release assay, Enzyme-LinkedImmunospot (ELIspot) assay, retrovirus production and transduction ofT-lymphocytes, and statistical analysis were as described in Example 1.

Test fixtures (hereinafter generically referred to as “G-Rex”) wereconstructed as shown in FIG. 6. Bottom 20 of each G-Rex 10 was comprisedof gas permeable silicone membrane, approximately 0.005 to 0.007 inchesthick. Pending U.S. Publication No. 2005/0106717 A1 to Wilson is amongmany other sources of information relating to the use of alternative gaspermeable materials and can be used to educate skilled artisans aboutgas permeable culture device shapes, features, and other usefulcharacteristics that are beneficial to many of the embodiments of thisinvention. In this Example 3, G-Rex (referred to as “G-Rex40”) had agrowth surface area of 10 cm², upon which a cell composition (shown asitem 30) rested, the characteristics of the cell composition variedthroughout the experiment as described within. Medium volume (shown asitem 40) unless otherwise indicated was 30 mL, creating a medium volumeto growth surface area ratio of 3 ml/cm².

Activated EBV-specific CTL and irradiated autologous EBV-LCLs at theconventional 4:1 ratio of CTL:LCL were cultured in G-Rex40 devices.EBV-CTLs were seeded at a surface density of 5×10⁵ cells/cm² in theG-Rex40 and the rate of EBV-CTL population expansion was compared withEBV-CTL seeded at the same surface density in a standard 24-well platewith a medium volume to growth surface area of 1 ml/cm². After 3 days,as shown in FIG. 7A (p=0.005), the EBV-CTLs in the G-Rex40 had increasedfrom 5×10⁵/cm² to a median of 7.9×10⁶/cm² (range 5.7 to 8.1×10⁶/cm²)without any medium exchange. In contrast, EBV-CTLs cultured 3 days inconventional 24-well plates only increased from a surface density of5×10⁵/cm² to a median of 1.8×10⁶/cm² (range 1.7 to 2.5×10⁶/cm²) by day3. In the G-Rex40, surface density could be further increased byreplenishing medium whereas cell surface density could not be increasedby replenishing medium or IL2 in the 24-well plate. For example, EBV-CTLsurface density further increased in the G-Rex40 to 9.5×10⁶ cells/cm²(range 8.5×10⁶to 11.0×10⁶/cm²) after replenishing the medium and IL-2 onday 7 (data not shown).

To understand the mechanism behind the superior cell expansion in theG-Rex device, we assessed the viability of OKT3-stimulated peripheralblood T cells using flow cytometric forward vs. side scatter analysis onday 5 of culture. EBV-CTLs could not be assessed in this assay due tothe presence of residual irradiated EBV-LCL in the cultures, which wouldinterfere with the analysis. As shown in FIG. 7B, cell viability wassignificantly higher in the G-Rex40 cultures was significantly higher(89.2% viability in the G-Rex40 vs. 49.9% viability in the 24-wellplate). We then analyzed the cultures each day for 7 days usingAnnexin-PI7AAD to distinguish between live and apoptotic/necrotic cells,and observed consistently lower viability in T-cells expanded in 24 wellplates compared to those in the G-Rex, as shown in FIG. 7C. These dataindicate the cumulative improved survival of proliferating cellscontributed to the increased cell numbers in the G-Rex devices comparedto the 24-well plates.

To determine if there was also a contribution from an increased numberof cell divisions in the G-Rex versus the 24-well plates, T-cells werelabeled with CFSE on day 0 and divided between a G-Rex40 device with a40 ml medium volume and a 24 well plate with each well at a 2 ml mediumvolume. Daily flow cytometric analysis demonstrated no differences inthe number of cell divisions from day 1 to day 3. From day 3 onwards,however, the population of desired cells cultured in the G-Rex40continued to increase at a rate that exceeded the diminishing rate ofthe 2 ml wells, indicating that the culture conditions had becomelimiting as shown in FIG. 7D. Thus, the large population of desiredcells in the G-Rex40 test fixtures resulted from a combination ofdecreased cell death and sustained proliferation relative toconventional methods.

EXAMPLE 6

By use of unconventionally high ratios of medium volume to growthsurface area and use of growth surfaces comprised of gas permeablematerial, the need to feed culture during production can be reducedwhile simultaneously obtaining unconventionally high desired cellsurface density.

This was demonstrated through use of G-Rex test fixtures for theinitiation and expansion of EBV:LCLs. For purposes of this example,G-Rex2000 refers to device as described in FIG. 8, the exception beingthe bottom is comprised of a 100 cm² growth surface area and a 2000 mlmedium volume capacity is available. EBV-LCLs were cultured in andexpand in the G-Rex2000 without changing the cell phenotype. EBV-LCLwere plated into a G-Rex2000 at a surface density of 1×10⁵ cells/cm²along with 1000 ml of complete RPMI medium to create a medium volume tosurface area ratio of 10 ml/cm². For comparison, EBV-LCL were platedinto a T175 flask at a surface density of 5×10⁵ cells/cm² along with 30ml of complete RPMI medium to create a medium volume to surface arearatio of about 0.18 ml/cm². As presented in FIG. 8A, the EBV-LCLcultured in G-Rex2000 expanded more than those in the T175 flask withoutrequiring any manipulation or media change. This culture condition didnot modify the final cell product as evaluated by Q-PCR for EBER and Bcell marker CD20 as presented in FIG. 8B and FIG. 8C.

EXAMPLE 7

When sufficient feeder and/or antigen cells are not present at the onsetof culture, desired cells may not expand. However, the cell compositioncan be altered to include an additional cell type acting as feeder cellsand/or antigen presenting cell to allow expansion.

FIG. 9 shows an illustrative example in which we experimentallydemonstrated that a very low cumulative surface density of desired cellsand antigen presenting cells (in this case AL-CTLs and LCLs cellscombining to create a cell composition with a surface density of 30,000cells/cm²) was unable to initiate outgrowth of the AL-CTL population.However, this same cell composition could be made to grow by alteringthe composition to include another cell type acting as a feeder cell. Inthis case we evaluated a feeder layer of three various forms ofirradiated K562 cells at a surface density of about 0.5×10⁶ cells/cm²and in all cases the population of AL-CTL expanded from the initial cellcomposition depicted in the first column of the histogram to move from asurface density of just 15,000 cells/cm² to a surface density of 4.0×10⁶cells/cm² over 14 days. We also demonstrated, as opposed to the additionof a third cell type, increasing the population of LCLs achieved similarfavorable results. The high surface density used for the LCL or K562 wasarbitrarily chosen to demonstrate that a very low population of desiredcells can be used to initiate growth when the cell composition includesan adequate number of feeder and/or antigen specific cells. When feedercells are in short supply, expensive, or cumbersome to prepare, reducingtheir surface density to below 0.5×10⁶ cells/cm² is recommended. Ingeneral, and as we have demonstrated, when antigen presenting cellsand/or feeder cells are in the cell composition, the additive surfacedensity of the antigen presenting cells and/or feeder cells and thedesired cells should preferably be at least about 0.125×10⁶ cells/cm² tocreate enough surface density in the cell composition to initiate theexpansion of the desired cell population. Also, to attain the continuedexpansion beyond standard surface density limits, the use of growthsurfaces comprised of gas permeable material was used in this examplealong with a medium volume to surface area ratio of 4 ml/cm².

EXAMPLE 8

Reduced desired cell surface densities, altered responder cell tostimulatory cell ratios, increased medium to growth surface area ratios,and periodic distribution of cells at a low surface density culture ontogrowth surfaces comprised of gas permeable material allow more desiredcells to be produced in a shorter period of time and simplifies theproduction process when compared to other methods.

To further evaluate our ability to simplify and shorten the productionof desired cells, we used G-Rex test fixtures for the initiation andexpansion of EBV-CTLs. For purposes of this example, G-Rex500 refers todevice as described in FIG. 6, the exception being the bottom iscomprised of a 100 cm² growth surface area and a 500 ml medium volumecapacity is available.

For the initial stage of EBV-CTL production, we seeded PBMCs in theG-Rex40 at a surface density of 1×10⁶/cm² (total=10⁷ PBMCs distributedover 10 cm² growth surface area of the G-Rex40) and stimulated them withEBV-LCL using a 40:1 ratio of PBMC:EBV-LCL. For CTL production, this40:1 ratio is preferable in the first stimulation to maintain theantigen-specificity of the responder T-cells. After the initial stage ofculture, a second stage was initiated on day 9, wherein 1×10⁷ responderT-cells were transferred from the G-Rex40 to a G-Rex500 test fixture. Toinitiate stage two of culture, 200 ml of CTL medium was placed in theG-Rex500, creating a medium volume to surface area ratio at the onset ofstage two of 2 ml/cm² medium height at 2.0 cm above the growth surfacearea. The surface density of desired cells at the onset of stage two was1×10⁵ CTL/cm² with antigen presenting cells at a surface density of5×10⁵ LCL/cm², thereby creating a non-conventional 1:5 ratio of desiredcells to antigen presenting cells. This stage two cell surface densityand R:S ratio produced consistent EBV-CTL expansion in all donorsscreened. Four days later (day 13), IL-2 (50 U/ml—final concentration)was added directly to the culture, as was 200 ml of fresh medium,bringing medium volume to surface area ratio to 4 ml/cm². On day 16, thecells were harvested and counted. The median surface density of CTLsobtained was 6.5×10⁶ per cm² (range 2.4×10⁶ to 3.5×10⁷).

Compared to conventional protocols, the use of growth surfaces comprisedof gas permeable material allows increased medium volume to surface arearatios (i.e. greater than 1 ml/cm²), lower cell surface densities (i.e.less than 0.5×10⁶/cm²), and altered ratios of responder to stimulatorcells (less than 4:1) to create a decrease in production time. FIG. 10Ashows the comparison of this G-Rex approach of Example 8 to the use ofconventional methods of Example 1 and the G-Rex approach described inExample 5. As shown, the conventional method needed 23 days to deliveras many desired cells as could be delivered in either G-Rex method inabout 10 days. After 23 days, the G-Rex approach of Example 8 was ableto produce 23.7 more desired cells than the G-Rex method of Example 5and 68.4 times more desired cells than the conventional method ofExample 1. Furthermore, the desired cells continued to divide until day27-30 without requiring additional antigen presenting cell stimulationprovided the cultures were split when cell surface density was greaterthan 7×10⁶/cm².

Although the CTLs could not be viewed clearly in the G-Rex using lightmicroscopy, clusters of CTLs could be visualized by eye or by invertedmicroscope and the appearance of the cells on days 9, 16, and 23 ofculture is shown in FIG. 10B. Culture in the G-Rex did not change thephenotype of the expanded cells as shown in FIG. 10C, with greater than90% of the cell composition being CD3+ cells (96.7±1.7 vs. 92.8±5.6;G-Rex vs. 24-well), which were predominantly CD8+ (62.2%±38.3 vs.75%±21.7). Evaluation of the activation markers CD25 and CD27, and thememory markers CD45RO, CD45RA, and CD62L, demonstrated no substantivedifferences between EBV-CTLs expanded under each culture condition. Theantigen specificity was also unaffected by the culture conditions, asmeasured by ELIspot and pentamer analysis. FIG. 10D shows arepresentative culture in which T-cells stimulated with EBV peptideepitopes from LMP1, LMP2, BZLF1 and EBNA1 and stained with HLA-A2-LMP2peptide pentamers staining showed similar frequencies ofpeptide-specific T-cells. Further, the expanded cells maintained theircytolytic activity and specificity and killed autologous EBV-LCL (62%±12vs. 57%±8 at a 20:1 E:T ratio; G-Rex vs. 24-well plate), with lowkilling of the HLA mismatched EBV-LCL (15%±5 vs. 12%±7 20:1 ratio) asevaluated by ⁵¹Cr release assays as shown in FIG. 10E.

Discussion of various novel methods for improved cell production forcell therapy: Examples 1-8 have been presented to demonstrate to skilledartisans how the use of various conditions including reduced surfacedensity of the desired cell population at the onset of a productioncycle, reduced surface density ratios between responder cells andstimulating cells, growth surfaces comprised of gas permeable materials,and/or increased medium volume to growth surface area ratios can be usedto expedite and simplify the production of cells for research andclinical application of cell therapy. Although Examples 1-8 were relatedto the production of antigen specific T cells, these novel cultureconditions can be applied to many important suspension cell types withclinical relevance (or required for pre-clinical proof of concept murinemodels) including regulatory T cells (Treg), natural killer cells (NK),tumor infiltrating lymphocytes (TIL), primary T lymphocytes, a widevariety of antigen specific cells, and many others (all of which canalso be genetically modified to improve their function, in-vivopersistence or safety). Cells can be expanded with feeder cells and/orantigen presenting cells that can include PBMC, PHA blast, OKT3 T, Bblast, LCLs and K562, (natural or genetically modified to express andantigen and/or epitope as well as co-stimulatory molecules such as41BBL, OX40, CD80, CD86, HLA, and many others) which may or may not bepulsed with peptide and/or a relevant antigen.

Unconventionally Low Initial Surface Density: One aspect of the presentinvention is the discovery that production time can be reduced relativeto conventional methods by the use of lower desired cell surfacedensity. In this manner, desired cells are able to have a greaternumerical difference between their minimum and maximum cell surfacedensities than conventional methods allow. Preferably, when the rate ofdesired cell population growth has begun to diminish, but the quantityof desired cells is not yet sufficient to terminate production, thedesired cells are re-distributed upon additional growth surfacescomprised of gas permeable material at low starting surface density onceagain.

To explain how our novel cell production methods that rely upon lowersurface density at the onset of any given culture stage can be applied,an example is now described. FIG. 11 shows a graphical representation ofexpansion of a desired cell population on a growth surface under theconventional scenario as compared to population expansion of the desiredcell type using one aspect of the present invention. In this novelmethod, the surface density of desired cells at the onset of aproduction stage is less than conventional surface density. In order tomake the advantages of this novel method the focus, this explanationdoes not describe the process of initially obtaining the desired cellpopulation. The ‘Day” of culture starts at “0” to allow skilled artisansto more easily determine the relative time advantages of this novelmethod. In this example, each production cycle of the conventionalmethod begins at a conventional surface density of 0.5×10⁶ desiredcells/cm² while each production cycle of this example begins at a muchlower and unconventional surface density of 0.125×10⁶ desired cells/cm².Thus, 4 times more surface area (i.e. 500,000/125,000) is required inthis example to initiate the culture of than the conventional methodsrequire. In this example, the desired cells of the conventional methodreaches a maximum surface density of 2×10⁶ cells/cm² in 14 days. Thus, 1cm² of growth area delivers 2×10⁶ cells/cm² which are thenre-distributed onto 4 cm² of growth area so that production can becontinued using the conventional starting density of 0.5×10⁶ cells/cm²(i.e. 4 cm² times 0.5×10⁶ cells=2×10⁶ cells). The cycle repeats foranother 14 days at which point maximum cell surface density is againreached, with each of the 4 cm² of growth surface area delivering2.0×10⁶ cells for a total of 8.0×10⁶ cells that are then distributedonto 16 cm² of growth area and the growth cycle repeats to deliver atotal of 32×10⁶ cells over 42 days.

The novel method depicted in FIG. 11, instead of using the conventionalmethod of depositing 500,000 desired cells onto 1 cm² at the onset ofproduction, distributes the 500,000 cells equally onto 4 cm² of growtharea to create at unconventionally low starting surface density of125,000 desired cells/cm² on Day 0. In example the novel method, as withthe conventional method, has its growth rate about to diminish on Day 7.Cells in the novel method are at a surface density of 1×10⁶ cells/cm².Thus, at the time point where growth rate is about to diminish, thisstage of culture has produced 4×10⁶ cells that are then re-distributedonto 32 cm² of growth area so that production in Stage 2 can becontinued using the starting surface density of 0.125×10⁶ cells/cm²(i.e. 32 cm² times 0.125×10⁶ cells=4×10⁶ cells). The cycle, or stage, ofproduction repeats for another 7 days to Day 14, at which point maximumcell surface density is again reached, with each of the 32 cm² of growthsurface area containing 1.0×10⁶ desired cells to yield a total of 32×10⁶cells in just 14 days. Note how at the end of each production cycle, aswith the conventional method, the novel method delivers a multiple ofthe finishing surface density divided by the starting surface density.However, by lowering starting cell surface density and completing eachstage of production before cells have entered a growth production timeis dramatically lowered. This example that describes how, by loweringthe desired cell surface density (in this case to 0.125×10⁶ cells/cm²)relative to conventional cell surface density, the same quantity ofdesired cells are delivered in just 33% of the time as the conventionalmethod (14 days vs. 42 days).

Although we quantified the advantages using a starting surface densityof 0.125×10⁶ cells/cm², skilled artisans should be aware that thisexample of the present invention demonstrates that any reduction belowconventional cell surface density will reduce production duration.Furthermore, skilled artisans will recognize that in this and othernovel methods presented herein, the rate of cell growth and point atwhich diminished cell growth occurs described is for illustrativepurposes only and the actual rates will vary in each application basedon a wide variety of conditions such as medium composition, cell type,and the like. Additionally, for a given application, skilled artisanswill recognize that the advantage of this aspect of the presentinvention is the production time reduction resulting from the reductionof cell surface density below that of conventional cell surface densityin any particular application, wherein the particular conventionalsurface density used in this illustrative example may vary fromapplication to application.

Thus, one aspect of the methods of the present invention when there is adesire to minimize the duration of production for a given quantity ofdesired cells that reside within a cell composition by use of reducedcell surface density is now described. Desired cells should be depositedupon a growth surface at an unconventionally low cell surface densitysuch that:

-   -   a. the desired cells are in the presence of antigen presenting        cells and/or feeder cells and with medium volume to surface area        ratio of up to 1 ml/cm² if the growth surface is not comprised        of gas permeable and up to 2 ml/cm² if the growth surface is        comprised of gas permeable, and    -   b. the preferred surface density conditions at the onset of a        production cycle being such that the target cell surface density        is preferably less than 0.5×10⁶ cells/cm² and more preferably        diminishing as described in FIG. 4, and    -   c. the surface density of the desired cells plus the surface        density of the antigen presenting cells and/or feeder cells is        preferably at least about 1.25×10⁵ cells/cm².

Based on the examples above, it is advisable for one to verify that theexpansion of the desired cell population does not become limited ifthere is an attempt to further reduce the surface density of the antigenpresenting cells and/or feeder cells below 1.25×10⁵ cells/cm². Weselected 1.25×10⁵ cells/cm² based on the goal of demonstrating thatoutgrowth of a population of desired cells at unconventionally lowdensity can be achieved when augmented by an adequate supply of antigenpresenting cells and/or feeder cells.

Use of growth surfaces comprised of gas permeable material and highermedium volume to growth surface area ratios can simplify and shortenproduction. Another aspect of the present invention is the discoverythat the use of growth surfaces comprised of gas permeable material andmedium volume to growth surface area ratios that exceed conventionalratios, and repeated cycles of production that increase the amount ofgrowth surface area used over time will reduce production duration.

An illustrative example is now presented to show how these conditionscan reduce the duration of production. FIG. 12 augments the discussionto show an example of the advantages that can be obtained by utilizing agrowth surface comprised of gas permeable material and anunconventionally high medium volume to growth surface area ratio beyond1 or 2 ml/cm². The discussion that follows is intended to demonstrate toskilled artisans how, by use of such a method, several options becomeavailable including reducing production time, reducing the amount ofgrowth surface area used, and/or reducing labor and contamination risk.Skilled artisans will recognize that FIG. 12 and associated discussionis merely an example, and does not limit the scope of this invention.

The cell composition containing the desired cell population in thisillustrative example is assumed to consume about 1 ml per “X” period oftime. FIG. 12 shows two production processes, labeled “conventionalmethod” and “novel method.” At the onset of growth, each process beginswith desired cells at a surface density of 0.5×10⁶/cm². However, thegrowth surface of in the novel method is comprised of gas permeablematerial and medium volume to surface area ratio is 2 ml/cm² as opposedto the conventional method of 1 ml/cm². In time period “X”, the desiredcell population of the conventional method has a reached a surfacedensity plateau of 2×10⁶/cm² and is depleted of nutrients while theadditional medium volume of the novel method has allowed growth tocontinue and desired cell surface density is 3×10⁶/cm². If the novelmethod continues, it reaches a surface density of 4×10⁶/cm². Thus, manybeneficial options accrue. The novel method can be terminated prior totime “X” with more cells produced than the conventional method, can beterminated at time “X” with about 1.5 times more cells produced than theconventional method, or can continue until the medium is depleted ofnutrients with 2 times many desired cells produced as the conventionalmethod in twice the time but without any need to handle the device forfeeding. In order for the conventional method to gather as many cells,the cells must be harvested and the process reinitiated, adding laborand possible contamination risk. Since cell therapy applicationstypically only are able to start with a fixed number of cells, theconventional method does not allow the option of simply increasingsurface area at the onset of production.

FIG. 13 continues the example of FIG. 12 to show how more than oneproduction cycle can be of further benefit. FIG. 13 shows a graphicalrepresentation of expansion of a desired cell population on a growthsurface under the conventional method as compared to populationexpansion of the desired cell type under one novel method of the presentinvention in which the surface density of the novel method exceedssurface density of the conventional method. In order to make thisembodiment the focus, this explanation does not describe the process ofobtaining the desired cell population. The ‘Day” of culture starts at“0” to allow skilled artisans to more easily determine the relative timeadvantages of this aspect of the invention. In this example, bothcultures are initiated using conventional desired cell surface densityof 0.5×10⁵ cells/cm² at “Day 0”. In this illustrative example, thegrowth surface of the conventional method is also comprised of gaspermeable material. However, the medium volume to growth surface ratioin the conventional method is 1 ml/cm² as opposed to 4 ml/cm² in thenovel method. As shown in FIG. 13, the desired cell population in theconventional method begins to diminish in growth rate when it is at asurface density of about 1.5×10⁶ cells/cm² in about 4 days and reaches amaximum surface density of 2×10⁶ cells/cm² in 14 days. At that point thedesired cell population is distributed to 4 cm² of growth area at asurface density of 0.5×10⁶/cm² in fresh medium at 1.0 ml/cm² and theproduction cycle begins again, reaching a surface density of 2×10⁶cells/cm² in another 14 days and delivering 8×10⁶ desired cells in 28days. By comparison, the desired cell population in the novel methodbegins to diminish in growth rate when it is at a surface density ofabout 3×10⁶ cells/cm² in roughly about 10 to 11 days and could reach amaximum surface density of 4×10⁶ cells/cm² in 28 days. However, toaccelerate production, the cycle ends when the desired cell populationis still in a high rate of growth. Thus, at about 10 to 11 days the3×10⁶ cells are re-distributed to 6 cm² of growth surface area at asurface density of 0.5×10⁶/cm² in fresh medium at 4.0 ml/cm² and theproduction cycle begins again, with the desired cell population reachinga surface density of 3×10⁶ cells/cm² in roughly another 10 to 11 daysand delivering 18×10⁶ desired cells around 21 days. Thus, in about 75%of the time, the novel method has produced over 2 times the number ofdesired cells as compared to the conventional method.

We have been able to obtain cell surface density in excess of 10×10⁶cells/cm² upon growth surfaces comprised of gas permeable material,demonstrating that the use of the high surface density aspect of ourinvention is not limited to the density described in this example.

Thus, another example of the methods of the present invention when thereis a desire to minimize the duration of production for a given quantityof desired cells that reside within a cell composition by use of reducedcell surface density is now described:

-   -   a. seeding the desired cells upon a growth surface area        comprised of gas permeable material and in the presence of        antigen presenting cells and/or feeder cells and with medium        volume to surface area ratio of at least 2 ml/cm², and    -   b. establishing the preferred surface density conditions at the        onset of a production cycle such that the target cell surface        density is within the conventional density of about 0.5×10⁶        cells/cm², and    -   c. allowing the desired cell population to expand beyond the        conventional surface density of about 2×10⁶ cells/cm², and    -   d. if more of the desired cells are wanted, redistributing the        desired cells to additional growth surface comprised of gas        permeable material and repeating steps a-d until enough desired        cells are obtained.

When using these novel methods, further benefits can be attained bycombining the attributes of initiating culture using unconventionallylow surface area, using novel surface density ratios of desired cellsand/or feeder cells, utilizing a growth surface area comprised of gaspermeable material, utilizing unconventionally high ratios of mediumvolume to growth surface area, and conducting production in cycles. Theconditions can be varied at any cycle of production to achieve thedesired outcomes, such as striking a balance between reduced productiontime, surface area utilization, feeding frequency, and the like.

FIG. 14 shows another novel method in which still further advantagesrelative to conventional methods are obtained. As with otherillustrative embodiments described herein, skilled artisans willrecognize that the description herein does not limit the scope of thisinvention, but instead acts to describe how to attain advantages ofimproved production efficiency. In this example, desired cells aredoubling weekly in conventional conditions. The ‘Day” of culture startsat “0” to allow skilled artisans to more easily determine the relativetime advantages of this embodiment. Also, issues previously describedrelated to feeder and/or antigen presenting cell surface density ratiosare not repeated to simplify this example. For illustrative purposes,assume a starting population of 500,000 desired cells with a doublingtime of 7 days in conventional conditions is present on “day 0”production. The conventional method begins with a surface density of0.5×10⁶ cells/cm² and a medium volume to surface area ratio of 1 ml/cm².As shown, when the population of the desired cells reaches a surfacedensity of 2×10⁶ cells/cm² the cells are distributed onto additionalsurface area at a surface density of 0.5×10⁶ cells/cm² and theproduction cycle begins anew. The novel method of this example beginswith a surface density of 0.06×10⁶ cells/cm², a growth surface areacomprised of gas permeable material, and a medium volume to surface arearatio of 6 ml/cm². As shown, when the population is nearing the start ofa growth plateau, cells are redistributed to more growth surface area.In this case, the population is determined to be reaching plateau fromnoting that plateau is initiated in the conventional method when cellsurface density approaches 1.5 times the medium volume to surface arearatio (i.e. about 1.5×10⁶ cells/ml). Thus, at a surface density of about4.5×10⁶ cells/cm² at about 9 days, cells are distributed onto 36 cm² ofgrowth surface area and the production cycle begins anew.

FIG. 15 tabulates a comparison of each production method depicted inFIG. 14, and extends to stages to demonstrate the power of the novelmethod, and why it is wise to adjust the production protocol at variousstages to fully capture the efficiency. Note that the novel methodoverpowers the conventional method after completing just the secondstage of the production cycle, delivering nearly 1.37 times more cellsin only about half the time with just 61% of the surface arearequirement. However, note how the third stage of the production cyclecreates a massive increase in cells and a corresponding increase insurface area. Thus, one should model the production cycles to anticipatehow to adjust the initial cell surface density and/or final cell surfacedensity throughout each cycle of the process to attain an optimal levelof efficiency for any given process.

As an example, FIG. 16 shows an example of how one could alter variablesin the novel method to gain efficiency as production progresses. Forexample, an increase in the starting surface density of cycle 3 from0.06 to 0.70 cell/cm² and a change to the final surface density from 4.5to 7.5 cells/cm² can be undertaken. Increasing the final surface densityis a matter of increasing the medium volume to surface area ratio beyondthe initial 6 ml/cm² to a greater number. The greater the medium volumeto surface area, the longer the cycle remains in rapid growth phase(i.e. the population expansion prior to plateau). In this case we haveallowed 5 extra days to complete the rapid growth phase and raised themedium volume to surface area ratio to about 8 ml/cm². So doing, in thisexample, allows over 3 trillion cells to be produced in 34 days with areasonable surface area. For example, we have fabricated and testeddevices with about 625 cm² of growth surface comprised of gas permeablematerial. This is clearly a superior approach to producing cells thanthe conventional method.

Thus, another preferred embodiment of the methods of the presentinvention when there is a desire to minimize the duration of productionfor a given quantity of desired cells that reside within a cellcomposition by use of reduced cell surface density is now described:

-   -   a. seeding the desired cells upon a growth surface area        comprised of gas permeable material and in the presence of        antigen presenting cells and/or feeder cells and with medium        volume to surface area ratio of at least 2 ml/cm², and    -   b. establishing the preferred surface density conditions at the        onset of a production cycle such that the target cell surface        density is less than the conventional density, preferably at        between about 0.5×10⁶ desired cells/cm² and about 3900 desired        cells/cm² and total number of desired cells and antigen        presenting cells and/or feeder cells being at least about        1.25×10⁵ cells/cm², and    -   c. allowing the desired cell population to expand beyond the        conventional surface density of about 2×10⁶ cells/cm², and    -   d. if more of the desired cells are wanted, redistributing the        desired cells to additional growth surface comprised of gas        permeable material and repeating steps a-d until enough desired        cells are obtained.

The present invention provides devices and methods of cell culture thatallow far superior cell production, particularly for the field ofAdoptive Cell Therapy. It allows a wide variety of benefits relative tostate of the art devices and methods including reducing the time neededto provide a given number of cells, greater fold expansion of a desiredcell population from an initial quantity of cells, the ability to reduceand even eliminate the frequency of medium exchange, simplified methodsof cytokine addition, the ability to reduce and even eliminate the needto count cells to determine their quantity, the ability to greatlyreduce the amount of medium that cells need to be separated from postculture, the ability to create a more effective population of cells thatare antigen specific, and the capacity to scale linearly.

EXAMPLE 9

More efficient methods of producing cells within a static gas permeableculture device by establishing novel culture conditions at the start ofthe culture process.

Static cell culture experiments were conducted in which K562 cells werecultured in test devices configured with a growth surface comprised ofgas permeable silicone material and with wall height that allowed 10 cmof medium to reside above the growth surface. The growth surface washeld in a substantially horizontal position with a growth surfacesupport as described more thoroughly in Wilson '717. Medium was placedin the test devices at a medium height of 10 cm beyond the growthsurface, establishing a medium volume to growth area ratio of 10 ml/cm².K562 cells were also introduced into the test devices and the deviceswere placed into a cell culture incubator at 37 C, 5% CO₂, and 95% R.H,whereby cells were allowed to gravitate to the growth surface. Themedium was not perfused or subjected to forced agitation and gas was notforced to flow past the growth surface, instead making contact with thegrowth surface by random motion of the ambient atmosphere.

FIG. 17A, FIG. 17B, FIG. 17C, FIG. 17D, and FIG. 17E show, forillustrative purposes, a representative spreadsheet of the experimentalconditions and typical results. Initial static culture conditionsestablished surface densities ranging between 1.0E+06 to 6.25E+04cells/cm², cell densities ranging from 1.0E+05 to 6.25E+03 cells/ml,with medium residing above the growth surface in all conditions at aconstant height of 10 cm and all medium being the same formulation withglucose concentration at 240 mg/dl. The initial state of static culturewas day 0 and cell counts and glucose concentration were assessed on day4, day 8, day 11.

FIG. 18 compares the fold expansion of the population increase relativeto the surface density of each of the experimental conditions detailedin FIG. 17A through FIG. 17E. Fold expansion of each condition wasdetermined by dividing the cell surface density on day 11 by the cellsurface density on day 0. In a series of evaluations where surfacedensity is at a low limit of 5.0E+05 cells/cm² and medium height is atthe upper limit of 2.0 cm, we concluded that the best fold expansion ofK562 in gas permeable bags was about 4.8 fold. Thus, dotted line 6 showstypical fold expansion in state of the art K562 production methods usinggas permeable bags. Each surface density condition established in ourexperiments created a population expansion that exceeded state of theart population expansion. Of note, the ability to increase the foldexpansion of the population of cells greatly increased as conditions ofinitial static culture surface density decreased to 0.125E+06, and thenfurther reductions were less advantageous although still far superior tostate of the art methods.

Other observations were made that we explored further, particularlyrelated to glucose being a potential surrogate measure of the number ofcells present at any given time and the ability to perform extendedculture without feeding.

EXAMPLE 10

Novel methods to determine the quantity cells in a population residingwithin a static gas permeable culture device without need of countingcells.

We observed that glucose depletion rates were consistently indicative ofthe number of cells in culture despite the culture medium residing in astatic state and (other than just routine handling of the device) notsubjected to mechanically forced mixing such as by perfusion, shaking,or stirring prior to sampling. This finding opens the door to furthersimplification in the field of Adoptive Cell Therapy. For example, theact of counting cells to determine how well a culture is progressing isone of many factors that make cell production for Adoptive Cell Therapyimpractical. The use of a surrogate measure in lieu of cell counts,combined with the inventive disclosures herein, brings even moresimplification to cell production.

We have discovered that it is possible to use glucose concentration ofthe culture as a as a surrogate indicator of the population of theculture. For cultures in which cells reside upon a growth surfacecomprised of a given type of gas permeable material, knowing the minimumtotal medium volume needed for the culture to reach maximum surfacedensity and the total reduction in glucose concentration needed for theculture to reach maximum surface density sets the stage for a surrogateprediction of the number of cells in the population of the culture.Equipped with that knowledge, one initiating culture (or a stage ofculture) would determine the baseline glucose concentration of medium,the baseline volume of medium, and would keep track of the volume ofmedium added to the culture prior to taking a measure of glucoseconcentration at the time of population estimation. The estimated numberof cells in the population is a function of the prorated total reductionin glucose concentration needed to reach maximum cell density multipliedby the prorated minimum medium volume needed to reach maximum surfacedensity and multiplied by the maximum surface density possible on thegrowth surface.

We applied this method to cultures described throughout variousdisclosures of the present invention, in which the growth surface ofexperimental devices was comprised of dimethyl silicone, between about0.006 to 0.0012 inches thick. A series of experiments were undertakenthat determined minimum volume of medium needed to allow the cells toreach maximum surface density and the corresponding total reduction inglucose concentration. The total reduction in glucose concentration wasabout 250 mg/dl for a variety of cultures with various cell typesincluding K562, LCL, and T cells. We were able to create formulaicrelationships that were predictive of cell number in culture as showbelow, where:

A=baseline glucose concentration of medium

B=measure of glucose concentration at the time of population estimation

C=total reduction in glucose concentration needed to reach maximumsurface density

D=baseline volume of medium

E=volume of medium added after baseline

F=minimum total medium volume needed to reach maximum surface density

G=maximum surface density

E=surface area of the growth surface

[(A−B)/C]×[(D+E)/F]×G×E=estimated number of cells in the culturepopulation in the device. Note that the prorated minimum medium volumecannot exceed 100%, since additional medium will not increase surfacedensity beyond the maximum capacity. For example, if a culture requires10 ml to reach maximum surface density and the baseline volume of mediumplus the volume of medium added exceeds 10 ml, one should use 100% asthe prorated minimum medium volume.

Note that the predictive formulas require knowledge of the cell cultureapplications maximum cell density (and/or maximum surface density) underconditions in which cells reside on a growth surface comprised of theparticular gas permeable material the artisan has selected. Experimentscan be undertaken to make that determination. For example, to determinethe maximum cell surface density of K562 cells upon a growth surfacecomprised of the gas permeable material in our experimental fixtures(dimethyl silicone as described previously), we increased medium heightuntil surface density could increase no more. The minimum volume ofmedium needed to support a maximum attainable surface density of K562 atabout 12.0E+06 cells/cm² was determined to be 10 ml with a correspondingtotal reduction in glucose concentration of 250 mg/ml.

Illustrative examples of how this information could be used to assessthe number of cells in K562 culture follow. For the first example,assume medium is not added after the onset of culture and theseconditions exist:

baseline medium volume=10 ml

baseline glucose concentration=475 mg/dl

glucose sample=300 mg/dl

surface area of the growth surface=100 cm²

Then the calculation would proceed as follows:

[(475 mg/dl−300 mg/dl)/250 mg/dl)×(10 ml+0 ml)/10 ml]×12E+06cells/cm²×100 cm²=840×10⁶ cells.

As another example, assume medium is added after the onset of cultureand these conditions exist:

baseline medium volume=6 ml

baseline glucose concentration=475 mg/dl

glucose sample=300 mg/dl

surface area of the growth surface=100 cm²

Then the calculation would proceed as follows:

[((475 mg/dl−300 mg/dl)/250 mg/dl)×(6 ml+2 ml)/10 ml]×12E+06cells/cm²×100 cm²=672×10⁶ cells.

As yet another example, assume medium is added after the onset ofculture and these conditions exist:

baseline medium volume=6 ml

baseline glucose concentration=475 mg/dl

glucose sample=300 mg/dl

surface area of the growth surface=100 cm²

Then, since total medium volume added to the culture exceeds the minimumtotal medium volume needed to reach maximum surface density, proratedminimum medium volume goes to 100% and therefore the prorated valueequals 1, and the calculation would proceed as follows:

[((475 mg/dl−300 mg/dl)/250 mg/dl)×(1)]×12E+06 cells/cm²×100 cm²=840×10⁶cells.

Skilled artisans should be aware that by predetermining the maximum celldensity in medium (cells/cm2) that the specific cell type(s) can attainwhen residing on the growth surface comprised of a particular type ofgas permeable material, an alternative formulaic relationship can beused to estimate the number of cells in the culture. In that case, theformulaic relationship would be a function of; (the prorated totalreduction in glucose concentration needed to reach maximum celldensity)×(the volume in medium at the onset of culture plus the volumeof medium added to the culture)×(maximum cell density). Be advised thatin the event that the cumulative volume of medium exceeds that of theminimum volume of medium needed to reach maximum surface density, theminimum volume of medium should be used in place of the cumulativevolume (as no extra medium volume will increase the surface densitybeyond its maximum).

To help understand the predictive capacity of the formulas, we includedthe predictions of the number of cells in culture on row 10 of eachspreadsheet (normalized by growth surface area) of conditions shown inFIG. 17A through FIG. 17E. Comparison of row 10 with the counted cellsof row 12 shows how the number of cells in culture at any given time canbe determined with a reasonable degree of certainty by use of glucose asopposed to cell counts. In fact, cell counts may not be as accurate dueto the inability to ensure the cells are mixed uniformly into the mediumprior to counting. Thus, it may be more beneficial to rely on glucosemeasures. In a preferred embodiment, cell counts would not be taken atleast for 4 days, more preferably for 5 days, more preferably for 6days, more preferably for 7 days, more preferably for 8 days, and evenmore preferably not until the culture was terminated.

More experiments were undertaken to determine if the formula dictatingthe relationship between glucose depletion and the number of live cellsin the device was accurate when glucose concentration at the onset ofcultures varied. Test fixtures were identical to those previouslydescribed. For illustrative purposes, FIG. 19A shows a representativespreadsheet of the experimental conditions and typical results for theculture of K562 cells under equivalent starting conditions except forthe glucose concentration, which was 240 mg/dl vs. 475 mg/dl at theonset of culture. Results are graphically depicted in FIG. 19B , FIG.19C, FIG. 19D, FIG. 19E, and FIG. 19F. Population growth by cell countand as predicted by glucose depletion was normalized for surfacedensity.

FIG. 19B shows the population expansion under each condition over a timeperiod of 11 days. The population growth rate differed slightly, butarrived at about the same number in 11 days. FIG. 19C shows the glucosedepletion rate in each culture condition. FIG. 19D shows the glucoseconsumption rate in each culture condition. FIG. 19E shows an overlay ofthe predicted value, using the formulaic calculation of cell number,versus the cell number as determined by manual counts for the cultureinitiated at a glucose concentration of 240 mg/dl. FIG. 19F shows anoverlay of the predicted value, using the formulaic calculation of cellnumber, versus the cell number as determined by manual counts for theculture initiated at a glucose concentration of 475 mg/dl. Note thepredictive capacity of the formulaic approach relative to the method ofmanual cell counts. This further demonstrates that various embodimentsof the present invention can be utilized in conjunction with a method ofreducing, or even eliminating, the frequency of cell counts in lieu of asurrogate measure of the concentration of solutes in the medium.

This is a particularly powerful advantage relative to cell counts whenone wishes to use the gas permeable devices described in Wilson '717 orWilson '176. Skilled artisans will recognize the challenge of gettingaccurate distribution of cells in such devices and the potential formiscounts due to poor cell distribution into the medium. Thus, asurrogate measure that only relies on a medium sample in lieu of actualcell counts is of great benefit in the field of Adoptive Cell Therapy.

Equipped with this knowledge, manufacturers of gas permeable devices,including those described in Wilson '717 or Wilson '176, could provide asimplified cell production process that can easily determine the numberof live cells in culture within a gas permeable device, absent the needto count cells, by providing a gas permeable cell culture deviceincluding a growth surface comprised of gas permeable material andproviding instructions and/or disseminating information relating to thedisclosures of the present invention.

EXAMPLE 11

Less complicated methods of producing cells within a static gaspermeable culture device by establishing novel conditions at the startof the culture process in order to limit feeding frequency in staticcultures.

We undertook a series of experiments to determine the ability to reducefeeding frequency by use of the novel methods disclosed within relativeto state of the art culture methods, which require feeding every two tothree days.

Experiments were conducted in devices that included growth surfaces withsurface areas comprised of gas permeable material and varying capacityfor medium height. The following description of an experiment thatcompared medium volume and feeding frequency are illustrative of ourfindings. K562 cells and medium were introduced into the devices andthey were placed into a cell culture incubator at 37 C, 5% CO₂, and 95%R.H, whereby cells were allowed to gravitate to the growth surface at asurface density of 0.125E+06 cells/cm², determined to be advantageousfor superior population fold expansion as previously described.

Medium resided at a height of 2.5 cm, 5.0 cm, 10.0 cm, or 15.0 cm abovethe growth surface, which was comprised of silicone and had a surfacearea of 100 cm². The growth surface was held in a substantiallyhorizontal position with a growth surface support as described morethoroughly in Wilson '717. Thus, experimental conditions included ratiosof medium volume to the surface area of growth surfaces at 2.5 ml/cm²,5.0 ml/cm², 10.0 ml/cm², and 15.0 ml/cm². Thus, initial cell density was0.05E+06 cells/ml, 0.025E+06 cells/ml, 0.0125E+06 cells/ml, and0.008E+06 cells/ml respectively.

No further medium was added to the 10.0 ml/cm² or 15.0 ml/cm²conditions. The original medium volume of the 2.5 ml/cm² condition wasdoubled by adding 2.5 ml/cm² of fresh medium on day 11, tripled on day14 by adding another 2.5 ml/cm² of fresh medium, and quadrupled on day17 by adding another 2.5 ml/cm² of fresh medium. The original mediumvolume of the 5.0 ml/cm² condition was doubled by adding 5.0 ml/cm² offresh medium on day 11. Eventually, the 2.5 ml/cm² and 5.0 ml/cm²conditions held 10.0 ml/cm² of [[fresh]] medium.

FIG. 20 shows a graphical representation of population growth,normalized for growth surface area, under various medium feedingconditions.

Note that all conditions eventually arrived at about the same number oflive cells. However, conditions that did not rely on the addition ofmedium during the culture arrived at the maximum number of live cellsfaster than the other conditions. For example, it took 20 days for the2.5 ml/cm² condition to arrive at maximum density while it only took 11days for the conditions that did not receive fresh medium after theonset of culture to arrive at the same maximum number of live cells.Also, the population growth rate was far superior in the unfedconditions. Also of importance, the condition that initiated culturewith medium at a height of 15.0 cm showed the capacity to maintain cellsin a prolonged duration of high viability relative to the 10.0 cmcondition. For example, viability was relatively high in the 15.0 cm fora period of about 4 days after the maximum cell population was attainedwhile it diminished rapidly after about 1 to 2 days in the 10.0 cmcondition. The practical benefit created here is a production processthat has a longer period of time in which to recover cells. Those ofordinary skill in the art in the field of Adoptive Cell Therapy willrecognize the value of this, as there are many reasons why one wouldderive value from a bigger window of time for cell recovery ranging froma delay in obtaining the results of quality control measures to changingconditions of the patient.

Skilled artisans will recognize that all of the experimental cultureconditions exhibited superior rates of cell population expansioncompared to state of the art methods for Adoptive Cell Therapy, butshould be aware that it is not only beneficial to reduce surface densityrelative to state of the art methods at the onset of culture, it isfurther possible to reduce the duration needed for production of desirednumber of cells by increasing medium height and/or medium to growth arearatios. Skilled artisans should recognize that improvements will beobtained in terms of the rate of population expansion as less surfacedensity and more medium height and/or a further increase in mediumvolume to growth surface area ratio is undertaken, and are encouraged tobalance the use of medium with the needs of the application. More mediumat the onset of culture can be provided if a larger window of time toharvest cells while they reside at high viability is sought. Of note,even if one were to start with a surface density at or above that ofstate of the art, such as at 2.0E+06 or greater, the process can besuperior since embodiments of the present invention can diminish feedingfrequency and reduce concerns about cell populations quickly losingviability. In general, a wide range of options have been demonstrated.For production of a population of cells with minimal feeding frequencyand shortened production duration, a most preferred initial culturecondition for production is a cell density of less than 0.5E+06cells/cm² and most preferably about 0.125E+06 cells/cm², and a mediumheight of about 5.0 cm or more and more preferably 10.0 cm to 15.0 cm,and/or a medium volume to growth surface area of about 5.0 ml/cm² ormore and more preferably 10.0 ml/cm² to 15.0 ml/cm², and/or an initialcell density about 0.025E+06 cells/ml or less and more preferably about0.0125E+06 cells/ml to about 0.008E+06 cells/ml.

EXAMPLE 12

Novel ways to limit feeding frequency of co-cultures residing within astatic gas permeable culture device and determine the size of the cellpopulation without need of counting cells, even though a portion of thecells are dying.

Adoptive Cell Therapy often relies on co-culture with cells that aredying because they were irradiated (such as APC's) or cells that aredying as a result of being removed from the body (such as PBMC's). Agood example of a co-culture application is in the culture of CMV-CTLs(cytomegalovirus specific cytotoxic T lymphocytes) out of a populationof PBMCs. Initially, the CMV-CTL population is a very small percentagerelative to the total population of PBMCs. As the culture progresses,the PBMC begin to die off and CMV-CTLs begin to grow. By the end ofculture, the frequency of CMV-CTL in the cell composition has increasedgreatly. The previously disclosed characteristics of the presentinvention, including those that contradict state of the art methods,such as reduced cell density, can be used to diminish feeding frequencyfor applications such as these.

We conducted static cell culture experiments to assess the ability ofglucose measurements to predict cell populations in the presence ofdying cells in co-cultures. Experimental devices included a growthsurface comprised of gas permeable silicone with a surface area of 100cm². The growth surface was comprised of silicone and held in asubstantially horizontal position with a growth surface support asdescribed more thoroughly in Wilson '717. FIG. 21 shows a spreadsheetthat summarizes conditions on day 0, day 9, and day 16. PBMCs mediumwere introduced into the experimental devices and the devices wereplaced into a cell culture incubator at 37 C, 5% CO₂, and 95% R.H,whereby cells were allowed to gravitate to the growth surface at asurface density of 5.0E+05 cells/cm². Medium resided at a height of 10.0cm and cell density was at 5.0E+04. Glucose measures were taken on day0, day 9, and day 16. Other than routine handling, the culture mediumand cells were not mixed by forced mixing with the aid of mechanicalequipment such as is the case with perfusion, shaking, or stirring. Row3 shows the increase in the percentage of antigen specific T cells(CMV-CTL) increases to about 27.9% of the population of the cellcomposition, and Row 4 shows how the CMV-CTL fold expansion as apercentage of the total population diminished after day 9. This isbecause the PBMC are dying. Row 19 demonstrates the ability of thesurrogate measures of solutes in the medium to predict the number ofcells in culture. Note that the predicted value is nearly identical tothe assessment of cell population by counting. Thus, the ability to usea surrogate measure to quantify cell population is useful even in cellcompositions in which components of the cell composition are dying.

EXAMPLE 13

Novel static gas permeable cell culture and cell recovery devices thatenable simplified methods of medium exchange and novel methods forgreatly diminishing the effort required to separate cells from mediumafter a cell production process is complete.

FIG. 22A shows a cross-sectional view of one example of an embodiment ofa present invention of cell culture and cell recovery device 1000configured to perform the disclosed novel cell culture and/or novel cellrecovery methods. Cell removal opening 1002 of cell removal conduit 1004resides in proximity of growth surface 1006. Medium removal opening 1008of medium removal conduit 1010 resides near growth surface 1006. Growthsurface 1006 is comprised of gas permeable material. There are manysources of information for skilled artisans to learn about appropriategas permeable material including Wilson '717. Preferably, growth surface1006 is liquid impermeable and non-porous. The distance from growthsurface 1006 to upper confine 1012 of internal volume 1014 defines thevolume of space where medium can reside. Although medium can reside inmedium removal conduit 1010 and cell removal conduit 1004, which canextend to a height beyond upper confine 1012, maximum medium heightshould be considered by skilled artisans to be the farthest distancefrom the bottom of internal volume 1014 to upper confine 1012 forpurposes of describing this embodiment. The cell culture and cellrecovery device does not require a stirring mechanism or any othermechanisms to mix the cells and/or medium.

FIG. 22B shows cell culture and cell recovery device 1000 in an initialstate of static culture at the onset of any given cell production stageof culture. Cell culture and cell recovery device 1000 resides in aposition in which growth surface 1006 is in oriented in a horizontalposition and cells 1016 have gravitated to growth surface 1006. In thisillustrative embodiment, growth surface support 1018 is used to holdgrowth surface 1006 in a horizontal position while allowing ambient gasto make contact with growth surface 1006 without need of pumps or othermechanisms to force gas past growth surface 1006. Skilled artisans canrefer to Wilson '717 for information about how to configure growthsurface support 1018. Although medium 1020 can reside at any levelwithin the confines of internal volume 1014, preferably the entireuppermost medium location 1022 is parallel to growth surface 1006 asshown. Cell culture and cell recovery device 1000 resides in anatmosphere suitable for cell culture and at a temperature suitable forcell culture. Ambient gas makes contact with gas permeable material ofgrowth surface 1006 by random motion and without need of pumps or othermechanisms to force gas to or from growth surface 1006.

Medium height is determined by the distance from the lowermost mediumlocation to the uppermost medium location, in this case the distancefrom growth surface 1006 to uppermost medium location 1022 at the onsetof culture being the initial static culture medium height. The ratio ofthe number of cells 1016 having gravitated to growth surface 1006 to thevolume of medium 1020 is an initial static culture cell density. Theratio of the number of cells 1016 upon growth surface 1006 to thesurface area of growth surface 1006 is the initial static culturesurface density. The ratio of medium 1020 volume to the surface area ofgrowth surface 1006 is an initial static culture medium volume to growthsurface area ratio. Cells reside in a state of static culture and theculture continues for a period of time. As described throughout thisdisclosure, the period of time may or may not include a mediumreplenishment step depending upon variables that include the initialstatic culture medium height, the initial static culture cell density,the initial static culture surface density, and/or the initial staticculture medium volume to growth surface area ratio.

FIG. 22C shows further steps to recover cells in a reduced volume ofmedium from cell culture and cell recovery device 1000. Medium isremoved by way of medium removal opening 1008 in medium removal conduit1010 while not withdrawing cells 1016. After this step, remaining mediumis shown as cell recovery medium 1024. The less cell recovery mediumthat remains, the less complicated the process of separating cells frommedium will become, which is inherent to state of the art methods forAdoptive Cell Therapy and currently relies on a great deal ofcentrifugation. However, it is critical that one take care not to lose asignificant amount of cells while reducing the medium volume.Preferably, fewer than 10% of cells are lost and more preferablyvirtually no cells are lost. For further guidance, we describe anexample of our use of this aspect of the present invention. In a gaspermeable device similar to that shown in FIG. 22A with a growth surfacecomprised of gas permeable silicone with a growth surface area of 100cm², a culture medium volume of 2000 ml and residing at a height at thepoint of medium volume reduction of 20 cm, thereby constituting a mediumvolume to growth surface area of 200 (ml/cm²), and a cultured cellpopulation of about 1 billion cells residing on the growth surface atthe point of medium reduction, we have demonstrated the ability to avoidvisible loss of cells while simultaneously obtaining a 100 foldreduction in medium volume and establishing a set of conditions at thepoint of cell recovery that were characterized by cell recovery mediumheight at a mere 0.2 cm (as determined when the growth surface was in ahorizontal position) and a cell recovery medium volume to growth surfacearea ratio at a mere 0.2 ml/cm². We then mixed the medium, in this caseby swirling the medium in the device, which readily lifted the cellsfrom the growth surface and which distributed them into the cellrecovery medium. We then removed the cells by way of a cell removalopening in a cell removal conduit. The cell removal opening was locatedalong the edge of the device and we tilted the device to allow medium tocollect at the location of the cell removal opening. Upon collection ofcells and cell recovery medium, we examined the cell concentration inthe cell recovery medium and it was striking, at about 50 million cellsper ml. Thus, we were able to concentrate the cells from an initial celldensity of about 0.5 million cells per ml by a factor of 100 without anyof the centrifugation equipment used in state of the art methods ofstatic cell culture in Adoptive Cell Therapy, leaving a mere fraction ofthe culture to be subjected to further processing for cell recovery. Inessence, we were able to reduce the volume of medium that needed to besubjected to centrifugation from 2000 ml to just 20 ml and the entireprocess took less than about 1 minute.

The location of the medium removal opening of the medium removal conduitis preferably located at a distance of 0.2 cm or more from the growthsurface when the growth surface resides in a horizontal position. Forexample, between 0.2 cm and 2.0 cm from the growth surface when thegrowth surface resides in a horizontal position allows significantvolume reduction for many of the cell culture methods of the presentinvention. The upper limit of the distance between the medium removalopening and the growth surface when the growth surface resides in ahorizontal position is preferably a distance that takes into account thetypical height of medium at the point where medium is to be decreasedfor cell recovery. For example, if one seeks to reduce the volume ofmedium that needs to be centrifuged by 50% relative to state of the artmethods of static cell culture, the medium removal opening of the mediumremoval conduit would be located at 50% of the medium height (assumingthe device was designed such that the medium resided entirely over thegrowth surface). Since use of laboratory space is at a premium, deviceheight should be about the height of medium expected to reside withinit. Therefore, to provide the option of getting at least a two-foldreduction in medium volume processing relative to state of the artmethods, a good rule of thumb is to design the device with a height thatis at or just beyond typical medium height during use and locate mediumremoval opening of the medium removal conduit at any location from about0.2 cm from the growth surface (when the growth surface resides in ahorizontal position) to about the halfway point from the top of thedevice to the growth surface as measured from the inside of the device.For example, if the distance from the upper confine of growth medium inthe device to the growth surface represents the potential height ofmedium in the device, the medium removal opening would preferably belocated 0.2 cm or more above the growth surface when the growth surfaceresides in a horizontal position and 50% or less of the potential mediumheight. In the event it is uncertain where the medium height willreside, more than one medium removal conduit could be present in thedevice.

The cell removal opening of the cell removal conduit is preferablylocated along the lower edge of the device and can collect cell recoverymedium without reorienting the device. However, the device can bereoriented if desired. FIG. 22D shows the process of reorienting cellculture and cell recovery device 1000 into a position at an angle 1026that deviates from the original horizontal cell culture position inorder to relocate cell recovery medium 1024, having cells 1016distributed within it, relative to cell removal opening 1002 of cellremoval conduit 1004, whereby cell recovery medium 1024 can subsequentlybe withdrawn.

However, the cell removal opening of the cell removal conduit need notbe located along the lower edge of the device. Once the step of mixingthe cells in the cell recovery medium is complete, the cell recoverymedium can be removed from any location in the device by simply rotatingthe device until the cell recovery medium is located at the cell removalopening, and then withdrawing the cell recovery medium by way of thecell removal conduit. Skilled artisans should recognize that theconduits need not be as shown, but can be any configuration. The keydesign feature is the ability to place the medium removal opening in thepreferred locations relative the growth surface as previously described.Thus, the conduits can be as simple as locating a septum in the side ofthe device or as complex as telescoping tubes. Also skilled artisansshould be aware that the method of cell culture and cell recovery neednot rely on closed system configurations, but can be practiced by simplemeans in open system configuration also. For example, we have conductedthe method and repeated the steps described above with an open systemdevice of the type described in Wilson '717 with use of a pipette as themedium removal conduit and as cell removal conduit while achieving theconcentrations described above.

To capture the advantages of increased cell culture medium volume togrowth surface area ratios described in various embodiments of thepresent invention, the internal height of the cell culture and cellrecovery device should preferably be at least more than 2.0 cm in anyparticular application. Also, to facilitate cell culture and cellrecovery, the cell culture and cell recovery device is preferablyconstructed with biocompatible materials, clear to allow visualassessment, and rigid to allow easy handling.

The discovery of a method for removing medium from the cell culture andcell recovery device of the present invention without removing cellscreates additional advantages relative to state of the art static cellculture methods for Adoptive Cell Therapy and are related to the mediumexchange process. Although the present disclosure describes novelmethods that avoid removal and replacement of medium in order toreplenish medium, there may be circumstances where an artisan may wishto perform that process. State of the art methods lead to cell removalwhen medium is removed and replaced and thus, the common practice is toremove medium, distribute it to one or more new devices, and add mediumto all the devices. Thus, more and more devices are present wheneverfeeding occurs. This need not occur with our novel methods, as the cellrecovery methods of our present invention leaves cells in the devicewhen medium is removed by use of a conduit that can be as simple as apipette. There is no need for the use of any screens, filters, orcentrifugation of the device to reduce the medium volume without cellloss. Medium can simply be removed and added to the same device untilthe cell population is at a maximum surface density.

In the case of medium removal and replacement, having already describedhow one can remove medium to a height of merely 0.2 cm without cellloss, it is easy to see now to perform medium exchange by simplyremoving medium to the desired height and/or volume by way of the mediumremoval opening of the medium removal conduit without cell removal, andthen adding medium to any volume or height desired.

Thus, equipped with this disclosure, a skilled artisan should seek tocreate a preferred embodiment of a static gas permeable cell culture andcell recovery device comprising:

-   -   a. a growth surface comprised of gas permeable material, and    -   b. the growth surface residing in a horizontal position when the        device is in operation, and    -   c. a medium removal conduit including a medium removal opening,        and    -   d. a cell removal conduit including a cell removal opening, and    -   e. an internal volume, and    -   f. an upper confine bounding the uppermost location of the        internal volume    -   g. the distance from the upper confine to the growth surface        being the potential medium height and the distance from the        upper confine to the growth surface being beyond 2.0 cm, and    -   h. the distance the medium removal conduit resides above the        growth surface at least 0.2 cm above the growth surface when the        growth surface resides in a horizontal position, and no more        than 50% beyond the potential medium height.

The device should preferably include relevant aspects of devicesdescribed in Wilson '717. Also, equipped with this knowledge,manufacturers of gas permeable devices, including those described inWilson '717, could facilitate more efficient methods of cell culture byproviding users with a cell culture and cell recovery device including agrowth surface comprised of gas permeable material, a medium removalconduit, a medium removal opening, a cell removal conduit, a cellremoval opening, and providing instructions and/or disseminatinginformation for:

-   -   a. adding cells and medium into the device, and    -   b. the cells being of a mammalian and of a non-adherent cell        type, and    -   c. placing the cell culture and cell recovery device in an        atmosphere suitable for cell culture and at a temperature        suitable for cell culture and with the growth surface being        oriented in a horizontal position and the cells residing upon        the growth surface, and    -   d. allowing the cells to gravitate to said growth surface, and    -   e. the ratio of the number of the cells having gravitated to the        growth surface to the volume of the medium being an initial        static culture cell density, and    -   f. the ratio of the number of the cells having gravitated to the        growth surface to the surface area of the growth surface being        an initial static culture surface density, and    -   g. the ratio of the medium volume to the surface area of the        growth surface being an initial static culture medium volume to        growth surface area ratio, and    -   h. the distance from the lowermost medium location to the        uppermost medium location being an initial static culture medium        height, and    -   i. allowing a period of time for cells to reside in a state of        static culture and further including steps for recovering cells        from the cell culture and cell recovery device comprising:    -   j. a pre cell recovery step comprising removing a portion of        medium by way of the medium removal opening in a cell removal        conduit and not withdrawing cells, the remaining volume of        medium in the device being a cell recovery medium volume, and    -   k. the distance from the uppermost location of the cell recovery        medium to the lowermost location of the cell recovery medium        when the growth surface resides in the horizontal position being        a cell recovery medium height, and    -   l. the ratio of the volume of the cell recovery medium to the        surface area of the growth surface being a cell recovery medium        volume to growth surface area ratio, and    -   m. the ratio of the cell recovery medium volume to the medium        volume being the medium reduction percentage, and a cell        recovery step comprising:    -   n. mixing the cells into the cell recovery medium, and    -   o. the ratio of the number of cells in the recovery medium to        the volume of the cell recovery medium being a recovered cell        density, and    -   p. removing the cells and the cell recovery medium from the cell        culture and cell recovery device by way of said cell removal        opening in a cell removal conduit.

Preferably, the cell recovery medium volume to growth surface area ratiois at least 0.2 ml/cm² and the medium reduction percentage being atleast 50%. Skilled artisans are advised that this method is capable ofutilizing any of the embodiments of the present invention including thedesired initial static culture cell density, initial static culturesurface density, initial static culture medium volume to growth surfacearea ratio, and/or initial static culture medium height that provideadvantages described herein. Also, skilled artisans are encouraged torecognize that the method includes use for islets.

EXAMPLE 14

Novel methods of using a static gas permeable culture device forsuperior production of CAR T cells.

Experiments were conducted in experimental devices that included growthsurfaces with surface areas comprised of gas permeable material andvarying capacity for medium height. The growth surface was comprised ofsilicone with a 100 cm² surface area and held in a substantiallyhorizontal position with a growth surface support as described morethoroughly in Wilson '717.

Three conditions for expansion of transduced antigen specific T cells(CAR T cells) were evaluated. Evaluation A included CAR T cells in thepresence of K652 APC cells and included medium height at 10 cm.Evaluation B included CAR T cells without the presence of K652 APC cellsand included medium height at 10 cm. Evaluation C cultured CART cells inaccordance with state of the art methods.

FIG. 23A shows the conditions of Evaluation A at the onset of cultureand as the culture progressed. Of note, the ratio of APC to CAR T cellsat the onset of culture was 2:1 and medium resided at a height of 10 cm.FIG. 23B shows the conditions of Evaluation B at the onset of cultureand as the culture progressed. Of note, APC were not present at theonset of culture and medium resided at a height of 10 cm. FIG. 23C showsthe conditions of Evaluation C at the onset of culture and as theculture progressed. Of note, the ratio of APC to CART cells at the onsetof culture was 2:1 and medium resided at a height of 2 cm.

Unlike state of the art cultures in the field of Adoptive Cell Therapy,cytokine stimulation is undertaken during medium exchange by addingcytokine (such as IL2) to the fresh medium. Thus, cytokine stimulationis simultaneous with medium exchange. However, as disclosed in variousembodiments of the present invention, feeding frequency is greatlyreduced and even eliminated. Thus, we also used Condition A andCondition B to evaluate the capacity to add cytokine in the absence ofmedium exchange. In lieu of medium exchange we simply added a bolus ofIL2 at the same frequency and at a quantity that brought the medium thesame quantity per ml of state of the art methods and did not subject themedium to forced mixing of any sort to distribute the IL2 within themedium.

FIG. 23D shows the total live cells in culture at various time points inthe culture. As can be seen, the number of total live cells of ConditionA were far superior to either of the alternative conditions. FIG.23E1-E3 show the percentage of CAR T cell expression at the onset ofculture and at the completion of culture. Histogram A representsCondition A, histogram B represents Condition B, and histogram Crepresents Conditions C. Condition B demonstrates the disadvantage ofnot providing APC in the culture at culture onset. When APC's wereprovided at the onset of culture, CAR expression improved from aninitial state of about 40% to a state of about 80% by the end ofculture. FIG. 23F shows the total fold expansion of CAR T cells duringculture. It is clear that Condition A was able to generate atremendously greater fold expansion than state of the art methods shownin Condition C.

Also of note, as shown in FIG. 23G, prediction of the live cellpopulation in Evaluation A was representative of cell population asdetermined by manual counts. Furthermore, the devices used forEvaluation A and Evaluation B were able to have medium withdrawn at theend of culture, using the methods previously disclosed, from a state of1000 ml to a state of 20 ml without cell loss.

Another important finding was related to the presence of APC in culture.Clearly, the T cells recovered from Condition A have a greater capacityto kill tumor cells that express the relevant antigen (PSCA) due to amore enhanced T cell product, which at the end of the culture has agreater percentage of CAR expressing T cells (CAR-PSCA) in thepopulation relative to its state at the onset of culture. By comparison,Condition B, due to its lack of APCs at the onset of culture, is unableto increase the percentage of CAR expressing T cells (CAR-P SCA) in thecell population at all over the culture period.

FIG. 23H shows the capacity of cell obtained from Condition A andCondition B to kill tumor cells expressing PSCA, and to avoid killingcells that do not express the PSCA antigen. The ratio of effector cellsto PSCA antigen expressing cells (Du145 and Capan1) or non-PSCA antigenexpressing cells (293T) was 40:1. Effector cells (i.e. CART cells) wereobtained from the cultures of Condition A and of Condition B at day 11of culture.

FIGS. 24A, 24B, 24C, and 24D summarize side by side comparisons of thepopulation expansion of CART cells specific to PSCA and Muc1 using theinitial culture conditions described for Condition A (the exceptionbeing the antigen expression of the APC was PSCA and Muc1 respectively).It can be seen that the novel initial conditions of the presentinvention were able to produce a far greater number of CAR T cells in ashorter period of time than state of the art conditions in conventionalculture ware. Skilled artisans will recognize the advantages are notlimited to CAR T cells recognizing PSCA or Muc1 antigens, but areapplicable to CAR T cells recognizing any antigen.

The capacity to produce more cells in a shorter time period, addcytokine without need of medium exchange or forced mixing of the medium,eliminate the need to feed the culture with fresh medium, avoid the needto count cells manually, and reduce the amount of medium the cellsreside in at the time of cell recovery to just 2% of the volume presentat the end of culture demonstrated the power of the present invention toovercome many of the problems inherent to state of the art methods forthe production cells for Adoptive Cell Therapy.

EXAMPLE 15

The methods of present invention are scalable in direct proportion tothe surface area of the growth surface.

We undertook experiments to assess scalability of the culture processesdescribed herein and in the parent case. The ability to move from asmall growth area to a large growth area without need of re-establishingprotocols to optimize the culture process is a powerful advantage. Todetermine if that were the case, we compared outcomes of K562 culturesinitiated on growth surfaces comprised of gas permeable silicone, withsurface areas of 10 cm², 100 cm², and 640 cm². Initial conditionsincluded a surface density of 0.125E+06 cells/cm² and a medium height of10 cm. No medium replenishment was undertaken after the onset ofculture.

FIG. 24E shows the population growth curves of three gas permeableculture devices with differing growth areas. Series 1 represents thelive cell expansion of culture in the 640 cm² device, series 2 the 100cm² device, and series 3 the 10 cm² device. FIG. 24F shows thepopulation growth curves of FIG. 24E curves normalized to surfacedensity and clearly demonstrates linear scalability.

Skilled artisans are encouraged to review Wilson '176 in the event thatthey seek to increase the growth surface area by scaling the culture inthe vertical direction and will recognize that many of the embodimentsof the present invention can be undertaken using devices described inWilson '176.

General description of preferred embodiments: For the purposes of thisdisclosure, growth surface is the area in a device upon which cellsreside and is comprised of gas permeable material. Gas permeablematerial can be any materials know to skilled artisans in the field ofcell culture and are preferably liquid impermeable and non-porous. Thedevices and culture methods of the present invention can function in theabsence of gas being forced past the growth surface that is comprised ofgas permeable material. These methods pertain to static cell culture.

Preferred cell types: In embodiments of the present invention, if theculture comprises a single cell type, the cells are preferably antigenpresenting cells, and more preferably LCL or K562. If the culturecomprises a co-culture, preferably it includes effector cells (i.e.desired or target cells) in combination with APC or feeder cells and mayor may not include beads. Beads may also be a substitute for APC orfeeder cells. If APC's are present, preferably they are professionalantigen presenting cells, and more preferably of the type K562 or LCL,and even more preferably are irradiated. If present, unless they areislets, effector cells are preferably derived from peripheral blood ormarrow, and more preferably are T cells, NK, Treg, TIL, or MIL. Ifeffector cells are T cells, preferably they are naturally occurringantigen specific T cells or transduced antigen specific T cells.

Preferred surface density: In embodiments of the present invention,cells reside upon a growth surface comprised of gas permeable materialand at a preferred surface density less than 0.5E+06. Skilled artisanswill recognize that the disclosure allows an analogue reduction insurface density from less than 0.5E+06 in order to increase ratepopulation expansion relative to state of the art methods in the fieldof Adoptive Cell Therapy, with more and more reduction being preferred.Thus, for example, we have demonstrated the rate of population expansionwith surface density of 0.25E+06, 0.125E+06, and 0.0625E+06 exceeds thatof state of the art methods. Thus, skilled artisans are encouraged torecognize that surface density need not be limited to just the statedvalues of our examples, the possibilities are not discrete values, butinstead are analogue. For example, those of ordinary skill in the artare advised that initiating culture at a surface density of 0.49E+06would allow improved “fold expansion” of the population relative toinitiating culture at surface density of 0.5E+06 even though we have notprovided an example with that particular surface density. Skilledartisans are thus advised to take the analogue interpretation of surfacedensities presented in the examples of the present invention and in theparent case, and are not limited to the discrete values presentedherein.

Preferred cell density: In embodiments of the present invention, cellsreside upon a growth surface comprised of gas permeable material and ata preferred cell to medium density of less than 0.5E+06. Skilledartisans will recognize that the disclosure allows an analogue reductionin cell to medium density from less than 0.5E+06 in order to decreasethe frequency of medium replenishment relative to state of the artmethods in the field of Adoptive Cell Therapy, with more and morereduction being preferred. Thus, for example, we have demonstrated howto reduce the frequency of medium replenishment be decreasing the cellto medium density from the 0.5E+06 cell/ml lower limit of state of theart methods, while simultaneously being able to maintain a cellpopulation that can expand at a rate that exceeds that of state of theart methods. Skilled artisans are encouraged to recognize that surfacedensity need not be limited to just the stated values of our examples,the possibilities are not discrete values, but instead are analogue.Preferably, skilled artisans should seek to reduce cell to mediumdensity below 0.5E+06 to any particular value they see fit given theattributes they wish to obtain. Therefore, although we describeadvantages of reducing cell to medium density with discrete cell tomedium density identification in various examples here and in thepresent case, the present invention is not limited to the discretenumbers presented herein.

Increased medium volume to growth surface area ratio: In embodiments ofthe present invention, cells reside upon a growth surface comprised ofgas permeable material and advantages accrue by increasing the ratio ofmedium volume to the surface area of the growth surface. Skilledartisans will recognize that the disclosure allows an analogue increasein the ratio of medium volume to the surface area of the growth surfaceorder to provide numerous advantages when combined with other elementsof the present invention such as reduced surface density. Therefore,although we describe these related advantages by use of examples thathave discrete values here and in the parent case, the present inventionis not limited to the discrete numbers presented herein, and those ofordinary skill in the art are encouraged to recognize the values, andcombinations of values, presented are guiding them to obtain thedescribed advantages by analogue interpretation of the values. Thus, thepresent invention is not limited to the discrete numbers presentedherein.

Increased medium height: In embodiments of the present invention, cellsreside upon a growth surface comprised of gas permeable material andadvantages accrue by increasing height of medium relative to state ofthe art methods. Skilled artisans will recognize that the disclosureallows an analogue increase in the height of medium in order to providenumerous advantages when combined with other elements of the presentinvention such as reduced surface density. Therefore, although wedescribe these related advantages by use of examples that have discretevalues here and in the parent case, the present invention is not limitedto the discrete numbers presented herein, and those of ordinary skill inthe art are encouraged to recognize the values, and combinations ofvalues, presented are guiding them to obtain the described advantages byanalogue interpretation of the values. Thus, the present invention isnot limited to the discrete numbers presented herein.

Surrogate measures of the rate of solute change in medium in lieu ofcounting cells: In embodiments of the present invention, cells resideupon a growth surface comprised of gas permeable material and advantagesaccrue by the ability to determine how many cells are in culture withouthaving to count cells. Skilled artisans will recognize that thedisclosure shows examples of how the decay in glucose concentrationprovides a measure of cell number in culture. Skilled artisans will alsorecognize that glucose is but one measurable substrate within mediumthat is utilized by cells, and that one of ordinary skill in the art,given the disclosure of this invention, could rely on the concentrationdepletion and/or increase of other substrates in the medium to indicatecell number, such as lactate. Thus, the inventive aspect of thisembodiment of the invention is the finding that measure of substrates instatic cultures that are initiated with any of the novel surfacedensities, cell densities, medium heights, and/or medium volume togrowth area conditions is a good way to determine the progress ofexpansion of a population of cells. Thus, the present invention is notlimited to a glucose substrate. Thus, the present invention is notlimited to the discrete numbers presented herein.

Removal of medium without cell loss: In embodiments of the presentinvention, cells reside upon a growth surface comprised of gas permeablematerial in various advantageous surface densities and can reside underan increased height of medium (and/or an increased medium volume togrowth surface area ratio) relative to state of the art methods.Advantages accrue by subsequently decreasing medium height (and/or adecreased medium volume to growth surface area ratio) absent cell lossrelative to state of the art methods. Skilled artisans will recognizethat the disclosure allows an improved method of medium exchange (inwhich more devices need not be added to the process) and/or an improvedmethod of cell recovery (in which a smaller volume of medium must beprocessed to recover cells). Although we describe these relatedadvantages by use of examples that have discrete values, the presentinvention is not limited to the discrete numbers presented herein, andthose of ordinary skill in the art are encouraged to recognize thevalues, and combinations of values, presented are guiding them to obtainthe described advantages by analogue interpretation of the values. Forexample, medium can reside at any height, preferably beyond 2.0 cm (suchas 2.1 cm, 2.5 cm, 6.08 cm, 10.0 cm and on). The medium removal openingof the medium removal conduit, preferably at 0.2 cm or more above thegrowth surface, can therefore preferably reside at any height of 0.2 cmand beyond so long as it resides below the medium height at the time ofmedium removal without cell loss, thereby diminishing cell separationfrom medium and/or not forcing users to move cells to additional devicesduring medium exchange. Thus, the present invention is not limited tothe discrete numbers presented herein.

Gas permeable culture devices: In embodiments of the present invention,we describe discoveries including the capacity for cultures to beinitiated at surface densities below the limits of conventional wisdom,minimal manipulation to provide cytokines, medium provision strategiesthat increase the growth rate of a population of cells relative to stateof the art methods and reduce production duration novel cell culturedevices, including improvement to methods of Wilson '717 and Wilson'176. Furthermore, device manufacturers and suppliers should recognizethat the provision of gas permeable devices, including those describedin Wilson '717 and Wilson '176, should include dissemination of novelmethods of the present invention and/or of the parent case to the usersof such devices by way of instructions, protocols, and the like nomatter the form (paper, electronic, website, etc.). Thus, the scope ofthe present invention includes the provision of instructions and/ordissemination of the methods of the present invention by manufacturersand/or suppliers of gas permeable devices.

The invention being thus described, it would be obvious that the samemay be varied in many ways by one of ordinary skill in the art havinghad the benefit of the present disclosure. Such variations are notregarded as a departure from the spirit and scope of the invention, andsuch modifications as would be obvious to one skilled in the art areintended to be included within the scope of the following claims andtheir equivalents.

Each of the applications, patents, and papers cited in this applicationand as well as in each document or reference cited in each of theapplications, patents, and papers (including during the prosecution ofeach issued patent; “application cited documents”), pending U.S.Publication Nos. 005/0106717 A1 and 2008/0227176 A1, and each of the PCTand foreign applications or patents corresponding to and/or claimingpriority from any of these applications and patents, and each of thedocuments cited or referenced in each of the application citeddocuments, are hereby expressly incorporated herein.

Any incorporation by reference of documents above is limited such thatno subject matter is incorporated that is contrary to the explicitdisclosure herein. Any incorporation by reference of documents above isfurther limited such that no claims included in the documents areincorporated by reference herein. Any incorporation by reference ofdocuments above is yet further limited such that any definitionsprovided in the documents are not incorporated by reference hereinunless expressly included herein.

For purposes of interpreting the claims for the present invention, it isexpressly intended that the provisions of Section 112, sixth paragraphof 35 U.S.C. are not to be invoked unless the specific terms “means for”or “step for” are recited in a claim.

Those skilled in the art will recognize that numerous modifications canbe made to this disclosure without departing from the spirit of theinventions described herein. Therefore, it is not intended to limit thebreadth of the invention to embodiments and examples described. Rather,the scope of the invention is to be interpreted by the appended claimsand their equivalents.

1.-20. (canceled)
 21. A method of increasing a ratio of the number ofcells on a growth surface relative to a volume of media in a gaspermeable cell culture and cell recovery device comprising: a gaspermeable cell culture and cell recovery device comprising an internalvolume bounded at least in part by a growth surface and an opposingupper confine, said growth surface comprised of non-porous liquidimpermeable gas permeable material, at least a portion of said gaspermeable material in contact with ambient gas by random motion, amedium removal conduit including a medium removal opening, a cellremoval conduit including a cell removal opening, an initial volume ofmedium within the device and a number of cells on the growth surface ina state of static cell culture, the ratio of the number of cells to theinitial volume of medium being an initial concentration of cells, andwhen the device is oriented in a static culture position wherein thegrowth surface is in a horizontal position and is located below saidopposing upper confine, said medium removal opening is closer to theupper confine than said cell removal opening, and performing a step ofdecreasing the initial volume of medium comprising removing a portion ofthe initial volume of medium by way of the medium removal opening tocreate a reduced volume of medium, thereby increasing the initialconcentration of cells.
 22. The method of claim 21 wherein said initialvolume of medium resides at an initial medium height defined by thedistance between the growth surface and the uppermost location of thevolume of medium, and said initial medium height is beyond 2.0 cm. 23.The method of claim 21 wherein the ratio of the initial volume of mediumto the growth surface area is from two and twenty milliliters per squarecentimeter of growth surface area.
 24. The method of claim 21 whereinthe device includes a growth surface support.
 25. The method of claim 21wherein said gas permeable material is comprised of silicone.
 26. Themethod of claim 21 including a step of removing the cells afterperforming the step of decreasing the initial volume of mediumcomprising mixing the cells into the reduced volume of medium andremoving the cells and the reduced volume of medium from the device byway of the cell removal opening of the cell removal conduit.
 27. Themethod of claim 26 including orienting the device into a cell recoveryposition wherein the cell removal opening is at a low point of the cellrecovery medium.
 28. The method of claim 21 wherein the cells include Tcells.
 29. The method of claim 21 wherein the cell removal opening islocated along a lower edge of the device.
 30. A method of increasing aratio of a number of cells on a growth surface relative to a volume ofmedia in a gas permeable cell culture and cell recovery devicecomprising: a cell culture compartment bounded by a growth surface andan opposing upper confine, said growth surface comprised of non-porousliquid impermeable gas permeable material, at least a portion of saidgas permeable material in contact with a growth surface support thatholds the gas permeable material in a horizontal position during cultureand allows-ambient gas to contact the underside of the gas permeablematerial without being forced into contact, a medium removal conduitincluding a medium removal opening, a cell removal conduit including acell removal opening that acts to withdraw media and cells from a lowpoint in the device, a vent, an initial volume of medium within thedevice and a number of cells reside upon said growth surface, wherein,the ratio of the number of cells to the initial volume of medium beingan initial concentration of cells, the distance from the growth surfaceto the uppermost medium location of the initial volume of medium beingan initial static medium height, and when the device is oriented in astatic culture position the growth surface is in a horizontal position,said medium removal opening is closer to the upper confine than saidcell removal opening, performing a step of decreasing the initial staticmedium height comprising removing a portion of the initial volume ofmedium by way of the medium removal opening to create a reduced volumeof medium and a reduced static medium height, thereby increasing theinitial concentration of cells and decreasing the initial static mediumheight.
 31. The method of claim 30 wherein the distance from said growthsurface to said medium removal opening is less than the distance fromthe lowermost medium location of the initial volume of medium to theuppermost medium location of the initial volume of medium.
 32. Themethod of claim 30 wherein the ratio of the initial volume of media tothe growth surface area is from two and twenty milliliters per squarecentimeter of growth area.
 33. The method of claim 30 wherein said gaspermeable material is comprised of silicone.
 34. The method of claim 30including a step of removing the cells after performing the step ofdecreasing the initial static medium height comprising mixing the cellsinto the reduced volume of medium and removing the cells and the reducedvolume of medium from the device by way of the cell removal opening ofthe cell removal conduit.
 35. The method of claim 34 including orientingthe device into a cell recovery position wherein the cell removalopening is at a low point of the reduced volume of medium.
 36. Themethod of claim 30 where the cells include T cells.
 37. The method ofclaim 30 wherein the device includes more than one medium removalopening, each medium removal opening residing at a different height fromsaid growth surface when the growth surface is in a horizontal position.38. The method of claim 30 wherein the cell removal opening is locatedalong a lower edge of the device.